Diagnosis and therapy of cancer using SGP28-related molecules

ABSTRACT

The present invention relates to methods and compositions for the diagnosis and therapy of prostate cancer which utilize isolated polynucleotides corresponding to the human SGP28 gene, proteins encoded by the SGP28 gene and fragments thereof, and antibodies capable of specifically recognizing and binding to SGP28 proteins.

This application claims the benefit of U.S. provisional patentapplication No. 60/162,610, filed Oct. 28, 1999, the entire contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention described herein relates to methods and compositions forthe diagnosis and therapy of cancer, including prostate cancer,utilizing isolated polynucleotides, polypeptides, antibodies, andrelated molecules that correspond to or are reactive with humanSGP28/CRISP-3.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy continue to be the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the management of this disease. Although the serum PSAassay has been a very useful tool, its specificity and general utilityis widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic disease progression, includingthe transition from androgen dependence to androgen independence and thedevelopment of metastatic lesions (Klein et al., 1997, Nat. Med. 3:402).More recently identified prostate cancer markers include PCTA-1 (Su etal., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate stem cellantigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:1735), and STEAP (Hubert et al., 1999, Proc. Natl. Acad. Sci. USA96:14523).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thediagnosis and therapy of prostate cancer. The methods of the inventionutilize isolated polynucleotides corresponding to human SGP28, proteinsencoded by the SGP28 gene and fragments thereof, and antibodies capableof specifically recognizing and binding to SGP28 proteins. The methodsof the invention are based, in part, on the molecular cloning of a genethat is identical to SGP28 and that is highly over-expressed in humanprostate cancers. The invention is further based on the discovery that,as determined by immunohistochemistry, very high levels of SGP28 proteinare expressed and secreted into the lumen of cancerous prostate glandsas well as in PIN, a non-invasive precancerous prostate lesion, and inbone and lymph node metastases. The expression profile of SGP28disclosed herein indicates that SGP28 provides a useful diagnosticmarker and/or therapeutic target for prostate cancer. Moreover, theexpression of SGP28 in PIN suggests that it may be a marker for earlydiagnosis of prostate cancer, a much needed improvement over what ispresently available using PSA. The expression pattern of SGP28 inindividual clinical specimens suggests that SGP28 can be used toidentify individual patients who will be more responsive to onetreatment modality versus another. In addition, SGP28 may serve as asurrogate marker for monitoring the efficacy of a prostate cancertherapeutic regimen. SGP28 molecules provide a particularly attractivemarker for use in in vivo imaging methods due to its expression in lymphnode and bone metastases.

SGP28 mRNA expression is restricted to the prostate and ovary, and ismarkedly up-regulated in prostate tumors. Expression of SGP28 in matchednormal prostate/tumor samples from advanced prostate cancer patients,using both mRNA and protein detection methods, shows a high degree ofup-regulated expression in the tumor tissue, suggesting that SGP28 is auseful marker for prostate cancer detection.

SGP28 is an extracellular soluble protein that has a predicted molecularweight of 29 kDa and a pI of 8.08. SGP28 has a signal peptide that iscleaved between amino acid residues 32 and 33, and includes twoextracellular protein SCP motifs (prosite domain PDOC00772), one atamino acids 150-160 and another at amino acids 170-182, both of SEQ IDNO: 3. The protein has strong homology to defensin proteins,particularly to beta-defensins, which are secreted products producedmainly by epithelial cells (O-Neil et al., 1999, J. Immunol.163:6718-24; Schroder et al., 1999, Int J. Biochem. Cell Biol.,31:645-51). As a defensin, the SGP28 protein may have the ability toinduce tumor cell death and/or may serve as a chemoattractant. SGP28 mayalso have a role in cell binding and/or in inducing cell growth.

A number of potential approaches to the treatment of prostate cancer andother cancers expressing SGP28 are described herein. The extracellularand soluble nature of this protein presents a number of therapeuticapproaches using molecules that target SGP28 and its function, as wellas molecules that target other proteins, factors and ligands thatinteract with SGP28. These therapeutic approaches include antibodytherapy with anti-SGP28 antibodies, small molecule therapies, andvaccine therapies. In addition, given its up-regulated expression inprostate cancer, SGP28 is useful as a diagnostic, staging and/orprognostic marker for prostate cancer and, similarly, may be a markerfor other cancers expressing this protein.

The invention provides polynucleotides corresponding or complementary toall or part of the SGP28 gene as described herein, mRNAs, and/or codingsequences, preferably in isolated form, including polynucleotidesencoding SGP28 proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid,and related molecules, polynucleotides or oligonucleotides complementaryto the SGP28 gene or mRNA sequences or parts thereof, andpolynucleotides or oligonucleotides which hybridize to the SGP28 gene,mRNAs, or to SGP28-encoding polynucleotides. Also provided are means forisolating cDNAs and the genes encoding SGP28. Recombinant DNA moleculescontaining SGP28 polynucleotides, cells transformed or transduced withsuch molecules, and host vector systems for the expression of SGP28 geneproducts are also provided.

The invention further provides SGP28 proteins and polypeptide fragmentsthereof, as well as antibodies that bind to SGP28 proteins andpolypeptide fragments thereof. The antibodies of the invention includepolyclonal and monoclonal antibodies, murine and other mammalianantibodies, chimeric antibodies, humanized and fully human antibodies,antibodies labeled with a detectable marker, and antibodies conjugatedto radionuclides, toxins or other therapeutic compositions.

The invention further provides methods for detecting the presence ofSGP28 polynucleotides and proteins in various biological samples, aswell as methods for identifying cells that express SGP28. The inventionfurther provides various therapeutic compositions and strategies,including particularly, antibody, vaccine and small molecule therapy,for treating cancers of the prostate.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C. Northern blot analysis of SGP28 mRNA expression in normaltissues (FIGS. 1A and 1B) and high level up-regulated expression inprostate cancer xenografts (Panel C). The lower molecular weight signalin normal testis is probably due to cross-hybridization of the probe(SSH fragment) to CRISP2/TPX-1 message. An identical transcript is seenfor CRISP2 on this normal panel using a gene specific oligonucleotideprobe as described by Kratzschmar et al., 1996, Eur J Biochem236(3):827-36. In FIG. 1A, lane 1 is heart, lane 2 is brain, lane 3 isplacenta, lane 4 is lung, lane 5 is liver, lane 6 is skeletal muscle,lane 7 is kidney, and lane 8 is pancreas. In FIG. 1B, lane 1 is spleen,lane 2 is thymus, lane 3 is prostate, lane 4 is testis, lane 5 is ovary,lane 6 is small intestine, lane 7 is colon, and lane 8 is leukocytes. InFIG. 1C, lane 1 is prostate, lane 2 is LAPC-4 AD, lane 3 is LAPC-4 AI,lane 4 is LAPC-9 AD, and lane 5 is LAPC-9 AI.

FIG. 2 Northern blot analysis of SGP28/36P1G3 mRNA expression in a panelof 3 prostate tumor (lanes 2, 4, 6) and normal adjacent tissue (lanes 1,3, 5) pairs, showing upregulation in 3 of the 3 tumor specimens.

FIG. 3. Western blot demonstrating that anti-SGP28 polyclonal antibodyidentifies SGP28 protein in LAPC4 and LAPC9 xenograft lysates and inLAPC4 cell line and transfected 293T cell line supernatants. Whole celllysates (WCL) and supernatants of LAPC4 cells and MYC/HIS SGP28transiently transfected 293T cells and LAPC4 and LAPC9 xenograft lysateswere subjected to western blotting using affinity purified rabbitanti-SGP28 pAb (1 μg/ml). SGP28 immunoreactive bands were visualized byincubation of the blots with HRP-conjugated anti-rabbit secondaryantibody, followed by enhanced chemiluminescence detection.

FIG. 4. Western blot analysis showing that anti-SGP28/CRISP-3 monoclonalantibody specifically detects SGP28/CRISP-3 protein in prostate cancercell lines and supernatants, prostate cancer xenografts, and clinicalprostate cancer tissue. Cell lysate and conditioned media from the LAPC4prostate cancer cell line and lysates from LAPC4 and LAPC9 prostatecancer xenografts and from a matched normal and cancerous prostateclinical specimen were separated by SDS-PAGE and transferred tonitrocellulose. The blot was then subjected to western analysis with a1:2 dilution of 4G6 anti-SGP28/CRISP-3 monoclonal antibody supernatant.Specific SGP28/CRISP-3 immunoreactive bands were then visualized byincubation with anti-mouse IgG-HRP conjugate secondary antibody anddevelopment with enhanced chemiluminescence and exposure toautoradiographic film.

FIG. 5A. Western blot analysis showing high level expression of SGP28 inprostate cancer clinical samples and LAPC xenografts. Matched clinicaltissue lysates of prostate cancer (PCa) and normal adjacent tissue (NAT)and of LAPC4 xenograft were subjected to western blotting with 1 μg/mlof affinity purified rabbit anti-SGP28 pAb. SGP28 immunoreactive bandswere visualized by incubation of the blots with HRP-conjugatedanti-rabbit secondary antibody followed by enhanced chemiluminescencedetection. Indicated with arrows is the SGP28/CRISP-3 immunoreactiveprotein doublet.

FIG. 5B. Western blot analysis showing high level expression of SGP28 inLAPC xenografts and low level expression in normal testis and lung.Normal tissue lysates of spleen, testis, kidney, liver and lung, andLAPC4 cell line and xenograft were subjected to western blotting asdescribed for FIG. 5A. Indicated with arrows is the SGP28/CRISP-3immunoreactive protein doublet.

FIG. 6A-B. Immunohistochemical analysis of SGP28 protein in Gleasonscore 7 prostate cancer (FIG. 6A) and high grade PIN (FIG. 6B), showinghigh level expression and secretion of SGP28 into the lumen of theprostate gland, using affinity purified polyclonal antibody. Strongstaining was observed in the epithelial cells of the prostate gland,especially at the lumenal borders. Staining was also observed within thelumen, indicating high level expression and secretion of SGP28 inprostate cancer and PIN.

FIGS. 7A-B. Immunohistochemical analysis demonstrating SGP28 proteinexpression in prostate cancer metastases to bone (FIG. 7A) and lymphnode (FIG. 7B).

FIG. 8A-D. Immunohistochemical analysis of SGP28 protein in prostatecancer and PIN. FIG. 8A shows immunohistochemical detection of SGP28 inprostate cancer at a magnification of 200×; FIG. 8B shows the same at800×. SGP28 expression in PIN is shown in FIG. 8C at 200×, and in FIG.8D at 800×.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for thediagnosis and therapy of prostate cancer which utilize isolatedpolynucleotides corresponding to the human SGP28 gene, proteins encodedby the SGP28 gene and fragments thereof, and antibodies capable ofspecifically recognizing and binding to SGP28 proteins. The invention isbased, in part, upon the isolation of a cDNA fragment corresponding tothe SGP28 gene by suppression subtraction hybridization cloning. ThiscDNA, designated 36P1G3, was sequenced and analyzed for homology toknown genes and ESTs in the major public databases. The 36P1G3 cDNAshowed identity to part of the reported sequence of the human SGP28gene. Primers designed to specifically amplify the gene corresponding to36P1G3 were then used to characterize SGP28 expression in prostatecancer xenografts, normal prostate, and a variety of other normaltissues. The nucleotide and amino acid sequences of SGP28 have beenreported (Kjeldsen et al., 1996, FEBS Lett. 380: 246-250; Kratzschmar etal., 1996, Eur J Biochem 236(3):827-36).

The expression profile of SGP28 suggests that it may represent an idealdiagnostic and therapeutic marker for prostate cancer. As determined bynorthern blot expression analysis, the expression of SGP28 mRNA innormal tissues is highly restricted to prostate, testis and ovary (FIG.1A-B). Very low level expression is detectable in pancreas (FIG. 1A).Interestingly, the prostate and ovary exhibit a 2.4 kb transcript, whiletestis expresses a 1.6 kb message (the 1.6 kb message could representanother SGP28 family member). Further, SGP28 mRNA expression is highlyupregulated in prostate cancer xenografts derived from advancedmetastatic stage disease (FIG. 1C). In addition, SGP28 protein isexpressed at high levels in these same prostate cancer xenografts aswell as in prostate cancer clinical specimens (FIG. 2). In matchednormal/cancerous prostate cancer clinical specimens, high levelover-expression of SGP28 protein relative to normal is detected,indicating that SGP28 provides an excellent diagnostic marker and/ortherapeutic target for prostate cancer. Immunohistochemical analysisestablishes that SGP28 protein is expressed and secreted at high levelsinto the lumen of cancerous and precancerous prostate glands, as well asin metastatic disease. Like PSA, SGP28 is secreted into the lumen andenters the serum in tissues where normal architecture is disturbed.

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers that have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers that have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least 10amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C., and most preferably to stringenthybridization conditions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium. citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative positions that are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions that are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. For example, % identity values may begenerated by WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996): http://blast.wustl/edu/blast/README.html). Furtherdetails regarding amino acid substitutions, which are consideredconservative under such criteria, are provided below.

Additional definitions are provided throughout the subsections thatfollow.

SGP28 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a SGP28 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a SGP28 protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a SGP28 gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides that hybridize to a SGP28 gene,mRNA, or to a SGP28 encoding polynucleotide (collectively, “SGP28polynucleotides”). As used herein, the SGP28 gene and protein is meantto include the SGP28 genes and proteins specifically described hereinand the genes and proteins corresponding to other SGP28 proteins andstructurally similar variants of the foregoing. Such other SGP28proteins and variants will generally have coding sequences that arehighly homologous to the SGP28 coding sequence, and preferably willshare at least about 50% amino acid identity and at least about 60%amino acid homology (using BLAST criteria), more preferably sharing 70%or greater homology (using BLAST criteria).

One embodiment of a SGP28 polynucleotide is a SGP28 polynucleotidehaving the sequence shown in Table 1 (SEQ ID NO: 2). A SGP28polynucleotide may comprise a polynucleotide having the nucleotidesequence of human SGP28 as shown in Table 1 (SEQ ID NO: 2), wherein Tcan also be U; a polynucleotide that encodes all or part of the SGP28protein; a sequence complementary to the foregoing; or a polynucleotidefragment of any of the foregoing. Another embodiment comprises apolynucleotide having the sequence as shown in Table 1 (SEQ ID NO: 2),from nucleotide residue number 3 through nucleotide residue number 776,wherein T can also be U.

Typical embodiments of the invention disclosed herein include SGP28polynucleotides encoding specific portions of the SGP28 mRNA sequencesuch as those that encode the protein and fragments thereof. Forexample, representative embodiments of the invention disclosed hereininclude: polynucleotides encoding about amino acid 1 to about amino acid10 of the SGP28 protein shown in Table 2 (SEQ ID NO: 3), polynucleotidesencoding about amino acid 20 to about amino acid 30 of the SGP28 proteinshown in Table 2 (SEQ ID NO: 3), polynucleotides encoding about aminoacid 30 to about amino acid 40 of the SGP28 protein shown in Table 2(SEQ ID NO: 3), polynucleotides encoding about amino acid 40 to aboutamino acid 50 of the SGP28 protein shown in Table 2 (SEQ ID NO: 3),polynucleotides encoding about amino acid 50 to about amino acid 60 ofthe SGP28 protein shown in Table 2 (SEQ ID NO: 3), polynucleotidesencoding about amino acid 60 to about amino acid 70 of the SGP28 proteinshown in Table 2 (SEQ ID NO: 3), polynucleotides encoding about aminoacid 70 to about amino acid 80 of the SGP28 protein shown in Table 2(SEQ ID NO: 3), polynucleotides encoding about amino acid 80 to aboutamino acid 90 of the SGP28 protein shown in Table 2 (SEQ ID NO: 3) andpolynucleotides encoding about amino acid 90 to about amino acid 100 ofthe SGP28 protein shown in Table 2 (SEQ ID NO: 3), etc. Following thisscheme, polynucleotides (of at least 10 amino acids) encoding portionsof the amino acid sequence of amino acids 100-258 of the SGP28 proteinshown in Table 2 (SEQ ID NO: 3) are typical embodiments of theinvention. Polynucleotides encoding larger portions of the SGP28 proteinare also contemplated. For example polynucleotides encoding from aboutamino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or40 or 50 etc.) of the SGP28 protein shown in Table 2 (SEQ ID NO: 3) maybe generated by a variety of techniques well known in the art.

Additional illustrative embodiments of the invention disclosed hereininclude SGP28 polynucleotide fragments encoding one or more of thebiological motifs contained within the SGP28 protein sequence. In oneembodiment, typical polynucleotide fragments of the invention can encodeone or more of the regions of SGP28 that exhibit homology tobeta-defensins. In another embodiment of the invention, typicalpolynucleotide fragments can encode one or more extracellular proteinsSCP/Tpx-1/Ag5/PR-1/Sc7 signature sequences. In yet another embodiment ofthe invention, typical polynucleotide fragments can encode sequencesthat are unique to one or more SGP28 alternative splicing variants.

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. As SGP28 is shown to be overexpressed inprostate cancer, these polynucleotides may be used in methods assessingthe status of SGP28 gene products in normal versus cancerous tissues.Typically, polynucleotides encoding specific regions of the SGP28protein may be used to assess the presence of perturbations (such asdeletions, insertions, point mutations etc.) in specific regions of theSGP28 gene products. Exemplary assays include both RT-PCR assays as wellas single-strand conformation polymorphism (SSCP) analysis (see e.g.Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of whichutilize polynucleotides encoding specific regions of a protein toexamine these regions within the protein. Assays and methods foranalyzing sequences to detect single nucleotide polymorphisms are alsoavailable (Irizarry, et al., 2000, Nature Genetics 26(2):223-236.

Other specifically contemplated embodiments of the invention disclosedherein are genomic DNA, cDNAs, ribozymes, and antisense molecules,including morpholino anti-sense molecules, as well as nucleic acidmolecules based on an alternative backbone or including alternativebases, whether derived from natural sources or synthesized. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the SGP28 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonudeotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,SGP28. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The SGP28 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention may beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); andIyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990), thedisclosures of which are fully incorporated by reference herein.Additional SGP28 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see e.g.Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6:169-175).

The SGP28 antisense oligonucleotides of the present invention typicallymay be RNA or DNA that is complementary to and stably hybridizes withthe first 100 N-terminal codons or last 100 C-terminal codons, oroverlapping with the ATG start site, of the SGP28 genome or thecorresponding mRNA. While absolute complementarity is not required, highdegrees of complementarity are preferred. Use of an oligonucleotidecomplementary to this region allows for the selective hybridization toSGP28 mRNA and not to mRNA specifying other regulatory subunits ofprotein kinase. Preferably, the SGP28 antisense oligonucleotides of thepresent invention are a 15 to 30-mer fragment of the antisense DNAmolecule having a sequence that hybridizes to SGP28 mRNA. Optionally,SGP28 antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 N-terminal codons and last 10C-terminal codons of SGP28. Alternatively, the antisense molecules aremodified to employ ribozymes in the inhibition of SGP28 expression. L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a SGP28 polynucleotide in a sample and as ameans for detecting a cell expressing a SGP28 protein. Examples of suchprobes include polypeptides comprising all or part of the human SGP28cDNA sequence shown in Table 1 (SEQ ID NO: 2). Examples of primer pairscapable of specifically amplifying SGP28 mRNAs are also described in theExamples that follow. An example of a primer pairs capable ofspecifically amplifying SGP28 mRNAs is: (SEQ ID NO: 4) 5′ - AGT TGC CTTTCC TAG CTC CAC TCT - 3′ (SEQ ID NO: 5) 5′ - TCC CTT TCC ATA CTC CAC TCTTTG - 3′As will be understood by the skilled artisan, a great many differentprimers and probes may be prepared based on the sequences provided inherein and used effectively to amplify and/or detect a SGP28 mRNA.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the SGP28 gene or that encodepolypeptides other than SGP28 gene product or fragments thereof. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated SGP28 polynucleotide.

The SGP28 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the SGP28 gene(s), mRNA(s), orfragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as tools for identifying moleculesthat inhibit calcium entry specifically into prostate cells; as codingsequences capable of directing the expression of SGP28 polypeptides; astools for modulating or inhibiting the expression of the SGP28 gene(s)and/or translation of the SGP28 transcript(s); and as therapeuticagents.

Molecular and Biochemical Features of SGP28

Specific granule protein 28 (SGP28) is a secreted molecule identifiedfrom and expressed in specific granules of human neutrophils (Kjeldsenet al., 1996, FEBS Lett. 380: 246-250). SGP28 is identical to theprotein known as Cysteine-rich secretory protein 3 (CRISP-3)(Kratzschmar et al., 1996, Eur. J. Biochem. 236:827-36). SGP28/CRISP-3(hereinafter referred to as SGP28) is a 29 kD protein of 258 amino acidscontaining a C-terminal cysteine rich sequence comprising 16 cysteineresidues conserved among several CRISP family member proteins. SGP28belongs to a family of cysteine rich secretory proteins present inhumans, rodents, and equine, comprising CRISP-1 (Brooks et al., 1986,Eur. J. Biochem. 161:13-18; Haendler et al., 1993, Endocrinology133:192-198; Kratzschmar et al., 1996, Eur. J. Biochem. 236:827-36),CRISP-2/TPX-1 (Kasahara et al., 1989, Genomics 5:527-534; Mizuki et al.,1992, Mamm. Genome 3:274-280) and CRISP-3/SGP28 (Haendler et al., 1993,Endocrinology 133:192-198; Schambony et al., 1998. Biochimica etBiophysica Acta 1387:206-216; Schwidetzky et al., 1995, Biochem. J.309:831-836).

Human SGP28 has been identified in granules of neutrophils and SGP28expression has been detected in salivary gland, pancreas, prostate,epididymis, ovary and colon (Kratzschmar et al., 1996, Eur. J. Biochem.236:827-36). Expression of murine CRISP family proteins have beendetected in B-cells, salivary and lacrimal glands, epididymis, testisand mucosal cells (Pfisterer et al., 1996, Mol. Cell. Biol.16:6160-6168; Haendler et al., 1999 J. Cell. Physiology 178:371-378,Haendler et al., 1997, Eur. J. Biochem. 250:440-446), and marine CRISP-2and CRISP-3 are androgen regulated (Haendler et al., 1999 J. Cell.Physiology 178:371-378, Haendler et al., 1997, Eur. J. Biochem.250:440-446). It has been suggested that SGP28 and other CRISP familymembers may have a role in non-specific innate immunity (Kjeldsen etal., 1996, FEBS Lett. 380: 246-250; Pfisterer et al., 1996, Mol. Cell.Biol. 16:6160-6168, Haendler et al., 1999 J. Cell Physiology178:371-378).

As is described further in the Examples that follow, the SGP28 gene andprotein have been characterized in a variety of ways. For example,analyses of nucleotide coding and amino acid sequences were conducted inorder to identify conserved structural elements within the SGP28sequence, topological features, post-translational modifications, andpotentially related molecules. Northern blot analyses of SGP28 mRNAexpression were conducted in order to establish the range of normal andcancerous tissues expressing the various SGP28 messages. Western blotand immunohistochemical analyses of SGP28 protein expression inexperimentally transfected and cancerous cells and tissues wereconducted to determine expression and secretion patterns. SGP28 has a pIof 8.08 and a calculated molecular weight of 29.0 kDa.

Several secreted proteins have been described in prostate cancer, anumber of which have been shown to participate in the process of tumorformation and progression (Inoue K., 2000; Clin. Cancer Res. 6:2104-19;Dow J K et al., 2000, Urology 55:800-6). As SGP28 is a secreted protein,one of its potential functions is to regulate the microenvironment ofprostate cancer and of metastatic disease. In order to test thispossibility, SGP28 can be expressed and purified as a recombinantGST-SGP28 or SGP28-Myc/His form. Purified recombinant-SGP28 (such asGST-SGP28 or SGP28-Myc/His) is then incubated with a variety of celltypes that recapitulate the environment of the prostate, includingprostate epithelial cells, prostate tumor cell lines, prostate stromalcells, prostate endothelial cells and prostate neuroendocrine cells. Inaddition, recombinant-SGP28 is also incubated with cells found atmetastatic sites, such as bone marrow cells and cells of the immunesystem. Binding of SGP28 to intact cells is detected by FACS analysisand by calorimetric assay. This analysis is valuable as it identifies acell population that binds and may respond to SGP28. In addition, theidentification of a target cell population may provide a means ofisolating and identifying SGP28 receptors.

SGP28 has a strong homology to defensin proteins, in particular toβ-defensins. Beta-defensins are secreted products mainly produced byepithelial cells (O'Neil DA et al, 1999, J. Immunol. 163:6718-24;Schroder J M, Harder J., 1999, Int. J. Biochem. Cell Biol. 31:645-51).Defensins play an important role in preventing infections andsafeguarding the immunity of epithelial tissues. In addition, the humanHNP1 defensin has been shown to induce the death of tumor cells invitro. Investigating the role of SGP28 in cell death, purifiedrecombinant-SGP28 is incubated with a variety of cell types listed aboveand analyzed for apoptotic activity using FACS analysis of Annexin Vstained cells. SGP28 may also function as a chemoattractant, as has beenshown for other defensin molecules (Yang D et al. Leukoc Biol.2000;68:9-14, Yang D et al. Science. 1999, 286(5439):525-8.). Using achemotactic assay, one can evaluate the effect of SGP28 on the migrationof various types of cells, including epithelial, stromal, endothelialcells as well as monocytes, lymphocytes and dendritic cells.

Isolation of SGP28-Encoding Nucleic Acid Molecules

The SGP28 cDNA sequences described herein enable the isolation of otherpolynucleotides encoding SGP28 gene product(s), as well as the isolationof polynucleotides encoding SGP28 gene product homologues, alternativelyspliced isoforms, allelic variants, and mutant forms of the SGP28 geneproduct. Various molecular cloning methods that can be employed toisolate full length cDNAs encoding a SGP28 gene are well known (See, forexample, Sambrook, J. et al. Molecular Cloning: A Laboratory Manual, 2dedition., Cold Spring Harbor Press, New York, 1989; Current Protocols inMolecular Biology. Ausubel et al., Eds., Wiley and Sons, 995). Forexample, lambda phage cloning methodologies may be convenientlyemployed, using commercially available cloning systems (e.g., Lambda ZAPExpress, Stratagene). Phage clones containing SGP28 gene cDNAs may beidentified by probing with labeled SGP28 cDNA or a fragment thereo. Forexample, in one embodiment, the SGP28 cDNA (Table 1; SEQ ID NO: 2) or aportion thereof can be synthesized and used as a probe to retrieveoverlapping and full length cDNAs corresponding to a SGP28 gene. TheSGP28 gene itself may be isolated by screening genomic DNA libraries,bacterial artificial chromosome libraries (BACs), yeast artificialchromosome libraries (YACs), and the like, with SGP28 DNA probes orprimers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga SGP28 polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YACs, BACs, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, for example, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a SGP28 polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 cell). Examples of suitablemammalian cells include various prostate cancer cell lines such LNCaP,PC-3, DU145, LAPC-4, TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of a SGP28 may be used to generate SGP28 proteins orfragments thereof using any number of host vector systems routinely usedand widely known in the art.

A wide range of host vector systems suitable for the expression of SGP28proteins or fragments thereof are available, see for example, Sambrooket al., 1989, supra; Current Protocols in Molecular Biology, 1995,supra). Preferred vectors for mammalian expression include but are notlimited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vectorpSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expressionvectors, SGP28 may be preferably expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1,3T3, PC-3, LNCaP and TsuPr1. The host vector systems of the inventionare useful for the production of a SGP28 protein or fragment thereof.Such host-vector systems may be employed to study the functionalproperties of SGP28 and SGP28 mutations.

Proteins encoded by the SGP28 genes, or by fragments thereof, will havea variety of uses, including but not limited to generating antibodiesand in methods for identifying ligands and other agents and cellularconstituents that bind to a SGP28 gene product. Antibodies raisedagainst a SGP28 protein or fragment thereof may be useful in diagnosticand prognostic assays, imaging methodologies (including, particularly,cancer imaging), and therapeutic methods in the management of humancancers characterized by expression of a SGP28 protein, including butnot limited to cancer of the prostate. Various immunological assaysuseful for the detection of SGP28 proteins are contemplated, includingbut not limited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), immunocytochemical methods, and the like. Such antibodies maybe labeled and used as immunological imaging reagents capable ofdetecting prostate cells (e.g., in radioscintigraphic imaging methods).SGP28 proteins may also be particularly useful in generating cancervaccines, as further described below.

SGP28 Proteins

Another aspect of the present invention provides SGP28 proteins andpolypeptide fragments thereof. The SGP28 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs to the extentthat such variants and homologs can be isolated/generated andcharacterized without undue experimentation following the methodsoutlined below. Fusion proteins that combine parts of different SGP28proteins or fragments thereof, as well as fusion proteins of a SGP28protein and a heterologous polypeptide, are also included. Such SGP28proteins will be collectively referred to as the SGP28 proteins, theproteins of the invention, or SGP28. As used herein, the term “SGP28polypeptide” refers to a polypeptide fragment or a SGP28 protein of atleast 10 amino acids, preferably at least 15 amino acids.

A specific embodiment of a SGP28 protein comprises a polypeptide havingthe amino acid sequence of human SGP28 as shown in Table 2 (SEQ ID NO:3), from amino acid residue number 1 through about amino acid residuenumber 258 as shown therein. Another specific embodiment of a SGP28protein comprises a polypeptide having the amino acid sequence of humanSGP28 as shown in Table 2 (SEQ ID NO: 3), from about amino acid residuenumber 33 through about amino acid residue number 258 as shown therein.A specific embodiment of a SGP28 fragment comprises a peptide selectedfrom the group comprising amino acids 1-32 of the SGP28 protein sequenceshown in Table 2 (SEQ ID NO: 3), or one or both of the extracellularprotein SCP motifs at amino acid residues 150-160 (VVGHYTQVVWY; SEQ IDNO: 6) and 170-182 (YYVCQYCPAGNW; SEQ ID NO:7).

In general, naturally occurring allelic variants of human SGP28 willshare a high degree of structural identity and homology (e.g., 90% ormore identity). Typically, allelic variants of the SGP28 proteins willcontain conservative amino acid substitutions within the SGP28 sequencesdescribed herein or will contain a substitution of an amino acid from acorresponding position in a SGP28 homologue. One class of SGP28 allelicvariants will be proteins that share a high degree of homology with atleast a small region of a particular SGP28 amino acid sequence, but willfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation insertion or frame shift.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently b interchangeable, as can alanine (A)and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

SGP28 proteins, including variants, comprise at least one epitope incommon with a SGP28 protein having the amino acid sequence of Table 2(SEQ ID NO: 3), such that an antibody that specifically binds to a SGP28protein or variant will also specifically bind to the SGP28 proteinhaving the amino acid sequence of Table 2 (SEQ ID NO: 3). One class ofSGP28 protein variants shares 90% or more identity with the amino acidsequence of Table 2 (SEQ ID NO: 3). A more specific class of SGP28protein variants comprises an extracellular protein SCP motif asdescribed above. Preferred SGP28 protein variants are capable ofexhibiting one or more of the defensin functions described herein,including, for example, the ability to induce tumor death or tochemoattract and/or induce migration of cells.

SGP28 proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physicalmechanical or chemical methods are employed to remove the SGP28 proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated SGP28 protein. A purified SGP28 proteinmolecule will be substantially free of other proteins or molecules thatimpair the binding of SGP28 to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a SGP28 protein include a purified SGP28 protein and afunctional soluble SGP28 protein. In one form, such functional, solubleSGP28 proteins or fragments thereof retain the ability to bind antibodyor other ligand.

The invention also provides SGP28 polypeptides comprising biologicallyactive fragments of the SGP28 amino acid sequence, such as a polypeptidecorresponding to part of the amino acid sequence for SGP28 as shown inTable 2 (SEQ ID NO: 3). Such polypeptides of the invention exhibitproperties of the SGP28 protein, such as the ability to elicit thegeneration of antibodies that specifically bind an epitope associatedwith the SGP28 protein.

Embodiments of the invention disclosed herein include a wide variety ofart accepted variants of SGP28 proteins such as polypeptides havingamino acid insertions, deletions and substitutions. SGP28 variants canbe made using methods known in the art such as site-directedmutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis [Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller etal., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis Wells etal., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal., Philos Trans. R. Soc. London SerA, 317:415 (1986)] or other knowntechniques can be performed on the cloned DNA to produce the SGP28variant DNA. Scanning amino acid analysis can also be employed toidentify one or more amino acids along a contiguous sequence. Among thepreferred scanning amino acids are relatively small, neutral aminoacids. Such amino acids include alanine, glycine, serine, and cysteine.Alanine is typically a preferred scanning amino acid among this groupbecause it eliminates the side-chain beyond the beta-carbon and is lesslikely to alter the main-chain conformation of the variant. Alanine isalso typically preferred because it is the most common amino acid.Further, it is frequently found in both buried and exposed positions[Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol.Biol., 150:1 (1976)]. If alanine substitution does not yield adequateamounts of variant, an isosteric amino acid can be used.

As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 258 amino acid sequence of theSGP28 protein shown in Table 2 (SEQ ID NO: 3). For example,representative embodiments of the invention disclosed herein includepolypeptides consisting of about amino acid 1 to about amino acid 10 ofthe SGP28 protein shown in Table 2 (SEQ ID NO: 3), polypeptidesconsisting of about amino acid 20 to about amino acid 30 of the SGP28protein shown in Table 2 (SEQ ID NO: 3), polypeptides consisting ofabout amino acid 30 to about amino acid 40 of the SGP28 protein shown inTable 2 (SEQ ID NO: 3), polypeptides consisting of about amino acid 40to about amino acid 50 of the SGP28 protein shown in Table 2 (SEQ ID NO:3), polypeptides consisting of about amino acid 50 to about amino acid60 of the SGP28 protein shown in Table 2 (SEQ ID NO: 3), polypeptidesconsisting of about amino acid 60 to about amino acid 70 of the SGP28protein shown in Table 2 (SEQ ID NO: 3), polypeptides consisting ofabout amino acid 70 to about amino acid 80 of the SGP28 protein shown inTable 2 (SEQ ID NO: 3), polypeptides consisting of,about amino acid 80to about amino acid 90 of the SGP28 protein shown in Table 2 (SEQ ID NO:3) and polypeptides consisting of about amino acid 90 to about aminoacid 100 of the SGP28 protein shown in Table 2 (SEQ ID NO: 3), etc.Following this scheme, polypeptides consisting of portions of the aminoacid sequence of amino acids 100-258 of the SGP28 protein are typicalembodiments of the invention. Polypeptides consisting of larger portionsof the SGP28 protein are also contemplated. For example polypeptidesconsisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the SGP28 protein shown in Table 2(SEQ ID NO: 3) may be generated by a variety of techniques well known inthe art.

Additional illustrative embodiments of the invention disclosed hereininclude SGP28 polypeptides containing the amino acid residues of one ormore of the biological motifs contained within the SGP28 polypeptidesequence as shown in Table 2 (SEQ ID NO: 3). SGP28 polypeptidescontaining one or more of these motifs or other select regions ofinterest described herein will typically include an additional 5 to 25or more amino acid residues of adjacent SGP28 protein sequence on one orboth sides of the selected motif(s). In one embodiment, typicalpolypeptides of the invention can contain one or more of the regions ofSGP28 that exhibit homology to defensins. In another embodiment, typicalpolypeptides of the invention can contain one or more of the SGP28N-glycosylation sites such as NCSN (SEQ ID NO: 8) at residues 252-255(numbering from first amino acid residue shown in SEQ ID NO: 3). Inanother embodiment, typical polypeptides of the invention can containone or more of the SGP28 protein kinase C phosphorylation sites such asSLK at residues 106-108 and/or 231-233. In another embodiment, typicalpolypeptides of the invention can contain one or more of the SGP28casein kinase II phosphorylation sites such as SWFD at residues 128-131(SEQ ID NO: 9) and/or SCPD at residues 206-209 (SEQ ID NO: 10). Inanother embodiment, typical polypeptides of the invention can containone or more of the tyrosine kinase phosphorylation sites such as KCGENLYat residues 108-114 (SEQ ID NO: 11). In another embodiment, typicalpolypeptides of the invention can contain one or more of theN-myristoylation sites such as GLLPSF at residues 26-31 (SEQ ID NO: 12),GCGNAY at residues 164-169 (SEQ ID NO: 13), GNWANR at residues 188-193(SEQ ID NO: 14), GAPCAS at residues 201-206 (SEQ ID NO: 15) and/orGLCTNG at residues 214-219 (SEQ ID NO: 16). In another embodiment,typical polypeptides of the invention can contain one or more of theextracellular protein SCP signature sequences, such as amino acidresidues 150-160 of SEQ ID NO: 3, and/or amino acid residues 179-190 ofSEQ ID NO: 3. In another embodiment, typical polypeptides of theinvention can contain one or more predicted HLA-A2 binding peptides suchas amino acids 2-10 (TLFPVLLFL; SEQ ID NO: 17), amino acids 6-14(VLLFLVAGL; SEQ ID NO: 18), amino acids 30-38 (ALLTTQTQV; SEQ ID NO:19), amino acids 142-150 (VVWYSSYLV; SEQ ID NO: 20), amino acids 222-230(TLTCKHQLV; SEQ ID NO: 21), amino acids 175-183 (GNWANRLYV; SEQ ID NO:22), amino acids 7-15 (LLFLVAGLL; SEQ ID NO: 23), amino acids 141-149(QVVWYSSYL; SEQ ID NO: 24), amino acids 134-142 (AVVGHYTQV; SEQ ID NO:25), and amino acids 211-219 (DLYSNCKSL; SEQ ID NO: 26) of SGP28.Related embodiments of these inventions include polypeptides containingcombinations of the different motifs discussed above with preferableembodiments being those that contain no insertions, deletions orsubstitutions either within the motifs or the intervening sequences ofthese polypeptides.

SGP28 polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human SGP28 proteins disclosedherein. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a polypeptide fragment of a SGP28protein. In this regard, the SGP28-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of SGP28proteins. SGP28 polypeptides are particularly useful in generating andcharacterizing domain specific antibodies (e.g., antibodies recognizingan extracellular or intracellular epitope of a SGP28 protein), inidentifying agents or cellular factors that bind to SGP28 or aparticular structural domain thereof, and in various therapeuticcontexts, including but not limited to cancer vaccines. SGP28polypeptides containing particularly interesting structures can bepredicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus -Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-SGP28 antibodies or in identifying cellular factors thatbind to SGP28.

In a specific embodiment described in the examples that follow, asecreted form of SGP28 may be conveniently expressed in 293T cellstransfected with a CMV-driven expression vector encoding SGP28 with aC-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen). The secretedHIS-tagged SGP28 in the culture media may be purified using a nickelcolumn and standard techniques. Alternatively, an AP-tag system may beused. Various constructs for expression of SGP28 are described in theexamples below.

Modifications of SGP28 such as covalent modifications are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of an SGP28 polypeptidewith an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the SGP28.Another type of covalent modification of the SGP28 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence SGP28 (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceSGP28. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present. Anothertype of covalent modification of SGP28 comprises linking the SGP28polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The SGP28 of the present invention may also be modified in a way to forma chimeric molecule comprising SGP28 fused to another, heterologouspolypeptide or amino acid sequence. In one embodiment, such a chimericmolecule comprises a fusion of the SGP28 with a polyhistidine epitopetag, which provides an epitope to which immobilized nickel canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the SGP28. In an alternative embodiment, thechimeric molecule may comprise a fusion of the SGP28 with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of an SGP28polypeptide in place of at least one variable region within an Igmolecule. In a particularly preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgG1 molecule. For the production of immunoglobulinfusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

SGP28 Antibodies

Another aspect of the invention provides antibodies that bind to SGP28proteins and polypeptides. The most preferred antibodies willselectively bind to a SGP28 protein and will not bind (or will bindweakly) to non-SGP28 proteins and polypeptides. Anti-SGP28 antibodiesthat are particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen-binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, an antibody fragment is defined as at leasta portion of the variable region of the immunoglobulin molecule thatbinds to its target, i.e., the antigen binding region.

For some applications, it may be desirable to generate antibodies thatspecifically react with a particular SGP28 protein and/or an epitopewithin a particular structural domain. For example, preferred antibodiesuseful for cancer therapy and diagnostic imaging purposes are thosewhich react with an epitope in an extracellular region of the SGP28protein as expressed in cancer cells. Such antibodies may be generatedby using the SGP28 proteins described herein, or using peptides derivedfrom predicted extracellular domains thereof, as an immunogen. In thisregard, with reference to the SGP28 protein sequence shown in FIG. 1,regions in the sequence amino-terminal to the transmembrane domain maybe selected and used to design appropriate immunogens and screeningreagents for raising and selecting extracellular-specific SGP28antibodies.

SGP28 antibodies of the invention may be particularly useful in prostatecancer therapeutic strategies, diagnostic and prognostic assays, andimaging methodologies. Similarly, such antibodies may be useful in thetreatment, diagnosis, and/or prognosis of other cancers, to the extentSGP28 is also expressed or overexpressed in other types of cancer. Theinvention provides various immunological assays useful for the detectionand quantification of SGP28 and mutant SGP28 proteins and polypeptides.Such assays generally comprise one or more SGP28 antibodies capable ofrecognizing and binding a SGP28 or mutant SGP28 protein, as appropriate,and may be performed within various immunological assay formats wellknown in the art, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), and the like. Inaddition, immunological imaging methods capable of detecting prostatecancer are also provided by the invention, including but limited toradioscintigraphic imaging methods using labeled SGP28 antibodies. Suchassays may be used clinically in the detection, monitoring, andprognosis of prostate cancer, particularly advanced prostate cancer.

SGP28 antibodies may also be used in methods for purifying SGP28 andmutant SGP28 proteins and polypeptides and for isolating SGP28homologues and related molecules. For example, in one embodiment, themethod of purifying a SGP28 protein comprises incubating a SGP28antibody, which has been coupled to a solid matrix, with a lysate orother solution containing SGP28 under conditions which permit the SGP28antibody to bind to SGP28; washing the solid matrix to eliminateimpurities; and eluting the SGP28 from the coupled antibody. Other usesof the SGP28 antibodies of the invention include generatinganti-idiotypic antibodies that mimic the SGP28 protein.

SGP28 antibodies may also be used therapeutically by, for example,modulating or inhibiting the biological activity of a SGP28 protein ortargeting and destroying cancer cells expressing a SGP28 protein.Antibody therapy of prostate and other cancers is more specificallydescribed in a separate subsection below.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a SGP28 protein, peptide, or fragment, in isolatedor immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press,Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring HarborPress, NY (1989)). Examples of protein immunogens include recombinantSGP28 (expressed in a baculovirus system, mammalian system, etc.), SGP28extracellular domain, AP-tagged SGP28, etc. In addition, fusion proteinsof SGP28 may also be used, such as a fusion of SGP28 with GST,maltose-binding protein (MBP), green fluorescent protein (GFP),HisMax-TOPO or MycHis (see Examples below).

In a particular embodiment, a GST fusion protein comprising all or mostof the open reading frame amino acid sequence of Table 2 (SEQ ID NO: 3)may be produced and used as an immunogen to generate appropriateantibodies. Cells expressing or overexpressing SGP28 may also be usedfor immunizations. Similarly, any cell engineered to express SGP28 maybe used. Such strategies may result in the production of monoclonalantibodies with enhanced capacities for recognizing endogenous SGP28.Another useful immunogen comprises SGP28 pep tides linked to the plasmamembrane of sheep red blood cells.

The amino acid sequence of SGP28 as shown in Table 2 (SEQ ID NO: 3) maybe used to select specific regions of the SGP28 protein for generatingantibodies. For example, hydrophobicity and hydrophilicity analyses ofthe SGP28 amino acid sequence may be used to identify hydrophilicregions in the SGP28 structure. Regions of the SGP28 protein that showimmunogenic structure, as well as other regions and domains, can readilybe identified using various other methods known in the art, such asChou-Fasman, Garnier Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. Peptides of SGP28 predicted to bind HLA-A2 maybe selected for the generation of antibodies. Such predicted HLA-A2binding peptides include, but are not limited to, amino acids 2-10(TLFPVLLFL; SEQ ID NO: 17), amino acids 6-14 (VLLFLVAGL; SEQ ID NO: 18),amino acids 30-38 (ALLTTQTQV; SEQ ID NO: 19), amino acids 142-150(VVWYSSYLV; SEQ ID NO: 20), amino acids 222-230 (TLTCKHQLV; SEQ ID NO:21), amino acids 175-183 (GNWANRLYV; SEQ ID NO: 22), amino acids 7-15(LLFLVAGLL; SEQ ID NO: 23), amino acids 141-149 (QVVWYSSYL; SEQ ID NO:24), amino acids 134-142 (AVVGHYTQV; SEQ ID NO: 25), and amino acids211-219 (DLYSNCKSL; SEQ ID NO: 26) of SGP28. As discussed in theexamples below, immunogenicity has been demonstrated with SGP28, whichwas used to generate polyclonal and monoclonal antibodies using rabbitsand mice, respectively. This B cell response (antibody production) isthe result of an initial T cell response elicited by the immunogenicportions of SGP28.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a SGP28 immunogen is conducted generally byinjection over a suitable period and with use of a suitable adjuvant, asis generally understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

SGP28 monoclonal antibodies are preferred and may be produced by variousmeans well known in the art. For example, immortalized cell lines whichsecrete a desired monoclonal antibody may be prepared using the standardhybridoma technology of Kohler and Milstein or modifications whichimmortalize producing B cells, a is generally known. The immortalizedcell lines secreting the desired antibodies are screened by immunoassayin which the antigen is the SGP28 protein or SGP28 fragment. When theappropriate immortalized cell culture secreting the desired antibody isidentified, the cells may be expanded and antibodies produced eitherfrom in vitro cultures or from ascites fluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the SGP28 protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.Humanized or human SGP28 antibodies may also be produced and arepreferred for use in therapeutic contexts. Methods for humanizing murineand other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al., 1986, Nature 321: 522-525;Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988,Science 239:1534-1536). See also, Carter et al., 1993, Proc. Nat'l Acad.Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296. Methodsfor producing fully human monoclonal antibodies include phage displayand transgenic animal technologies (for review, see Vaughan et al.,1998, Nature Biotechnology 16: 535-539).

Fully human SGP28 monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human SGP28 monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationW098/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of SGP28 antibodies with a SGP28 protein may be establishedby a number of well known means, including western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,SGP28 proteins, peptides, SGP28 expressing cells or extracts thereof.

A SGP28 antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule, such as acytotoxin or other therapeutic agent, and used for targeting the secondmolecule to a SGP28 positive cell (Vitetta, E. S. et al., 1993,Immunotoxin therapy, in DeVita, Jr., V. T. et al., eds., Cancer:Principles and Practice of Oncology, 4th ed., J. B. Lippincott Co.,Philadelphia, 2624-2636). Examples of cytotoxic agents include, but arenot limited to ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes such as ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y, and 1⁸⁶Re. Suitable detectable markers include, but are notlimited to, a radioisotope, a fluorescent compound, a bioluminescentcompound, chemiluminescent compound, a metal chelator or an enzyme.Antibodies may also be conjugated to an anti-cancer pro-drug activatingenzyme capable of converting the pro-drug to its active form. See, forexample, U.S. Pat. No. 4,975,287.

Further, bi-specific antibodies specific for two or more SGP28 epitopesmay be generated using methods generally known in the art. Further,antibody effector functions may be modified to enhance the therapeuticeffect of SGP28 antibodies on cancer cells. For example, cysteineresidues may be engineered into the Fc region, permitting the formationof interchain disulfide bonds and the generation of homodimers which mayhave enhanced capacities for internalization, ADCC and/or complementmediated cell killing (see, for example, Caron et al., 1992, J. Exp.Med. 176: 1191-1195; Shopes, 1992, J. Immunol. 148: 2918-2922).Homodimeric antibodies may also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

SGP28 Transgenic Animals

Nucleic acids that encode SGP28 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA that is integrated into the genomeof a cell from which a transgenic animal develops. In one embodiment,cDNA encoding SGP28 can be used to clone genomic DNA encoding SGP28 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells that express DNA encodingSGP28.

Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for SGP28 transgene incorporationwith tissue-specific enhancers. Transgenic animals that include a copyof a transgene encoding SGP28 introduced into the germ line of theanimal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding SGP28. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of SGP28 can be used to construct aSGP28 “knock out” animal that has a defective or altered gene encodingSGP28 as a result of homologous recombination between the endogenousgene encoding SGP28 and altered genomic DNA encoding SGP28 introducedinto an embryonic cell of the animal. For example, cDNA encoding SGP28can be used to clone genomic DNA encoding SGP28 in accordance withestablished techniques. A portion of the genomic DNA encoding SGP28 canbe deleted or replaced with another gene, such as a gene encoding aselectable marker that can be used to monitor integration.

Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector (see e.g., Thomas and Capecchi,1987, Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected (see e.g.,Li et al., 1992, Cell 69:915). The selected cells are then injected intoa blastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras (see e.g., Bradley, in Teratocarinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, 1987,pp.113-152).

A chimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the SGP28polypeptide.

Assays for Circulating and Excreted SGP28

Based on applicants immunohistochemical evidence of high level lumenalexpression of SGP28 in cancerous and precancerous prostate glands, it isexpected that prostate tumors would secrete SGP28 into the vasculatureand/or excrete SGP28 into urine or semen, where the protein may bedetected and quantified using assays and techniques well known in themolecular diagnostic field. Detecting and quantifying the levels ofcirculating or excreted SGP28 is expected to have a number of uses inthe diagnosis, staging, and prognosis of prostate cancer. A number ofdifferent technical approaches for the detection and quantification ofproteins in serum, urine or semen are well known in the art. BecauseSGP28 is a secreted protein expressed in cancers of the prostate andcolon, and possibly other cancers, assays for detecting and quantifyingSGP28 in blood or serum are expected to be useful for the detection,diagnosis, prognosis, and/or staging of a SGP28 expressing tumor in anindividual. For example, SGP28 mRNA expression in normal tissues isfound predominantly in prostate and ovary. However, high level proteinexpression is detected in prostate cancer as well as PIN. Accordingly,detection of serum SGP28 protein may provide an indication of thepresence of a prostatic tumor. Diagnosis of cancer may be made on thebasis of this information and/or other information. In respect ofprostate cancer, for example, such other information may include serumPSA measurements, DRE and/or ultrasonography. Further, the level ofSGP28 detected in the serum may provide information useful in staging orprognosis. For example, very high levels of SGP28 protein in serum maysuggest a larger and/or more aggressive tumor.

In addition, peripheral blood may be conveniently assayed for thepresence of SGP28 protein and/or SGP28 expressing cancer cells,including but not limited to prostate cancer, using RT-PCR to detectSGP28 expression. The presence of RT-PCR amplifiable SGP28 mRNA providesan indication of the presence of the cancer. RT-PCR detection assays fortumor cells in peripheral blood are currently being evaluated for use inthe diagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston etal., 1995, Clin. Chem. 41: 1687-1688). RT-PCR assays are well known inthe art.

In one embodiment, a capture ELISA is used to detect and quantify SGP28in serum, urine or semen. A capture ELISA for SGP28 comprises,generally, at least two monoclonal antibodies of different isotypes thatrecognize distinct epitopes of the SGP28 protein, or one anti-SGP28monoclonal antibody and a specific polyclonal serum derived from adifferent species (e.g., rabbit, goat, sheep, hamster, etc.). In thisassay, one reagent serves as the capture (or coating) antibody and theother as the detection antibody.

As discussed in detail below, levels of SGP28 including SGP28 serumlevels may be used to provide an indication of the presence, extent andaggressiveness of a SGP28 expressing tumor. As noted, above SGP28 sharesa number of characteristics with PSA which is the most important,accurate, and clinically useful biochemical marker in the prostate. Anyprocess that disrupts the normal architecture of the prostate allowsdiffusion of PSA into the stroma and microvasculature. Consequently,clinically important increases in serum prostate-specific antigen levelsare seen with prostatic cancers. In particular, the greater number ofmalignant cells and the stromal disruption associated with canceraccount for the increased serum prostate-specific antigen level. In thiscontext, serum prostate-specific antigen levels correlate positivelywith clinical stage, tumor volume, histologic grade, and the presence ofcapsular perforation and seminal vesicle invasion. See e.g. Bostwick, D.G., 1994, Am. J. Clin. Pathol. 102(4 Suppl 1): S31-S37.

Using PSA as the best analogous molecule, it is likely that becauseSGP28 is also a secreted molecule that exhibits a restricted pattern oftissue expression (including the prostate), the increasing load ofmalignant cells and the stromal disruption that occurs with cancer willmake the serum SGP28 antigen levels correlate positively with one ormore clinically relevant factors such as clinical stage, tumor volume,histologic grade, and the presence of capsular perforation and seminalvesicle invasion. Serum SGP28 measurements over time would be expectedto provide further information, wherein an increase in SGP28 would beexpected to reflect progression and the rate of the increase would beexpected to correlate with aggressiveness. Similary, a decline in serumSGP28 would be expected to reflect a slower growing or regressing tumor.The identification of SGP28 in serum may be useful to detect tumorinitiation and early stage disease. In patients who have undergonesurgery or therapy, serum SGP28 levels would be useful for monitoringtreatment response and potential recurrence.

Monitoring the Status of SGP28 and its Products

Assays that evaluate the status of the SGP28 gene and SGP28 geneproducts in an individual may provide information on the growth oroncogenic potential of a biological sample from this individual. Forexample, because SGP28 mRNA is so highly expressed in prostate cancers,and not in most normal tissue, assays that evaluate the relative levelsof SGP28 mRNA transcripts or proteins in a biological sample may be usedto diagnose a disease associated with SGP28 dysregulation, such ascancer, and may provide prognostic information useful in definingappropriate therapeutic options. Similarly, assays that evaluate theintegrity SGP28 nucleotide and amino acid sequences in a biologicalsample, may also be used in this context.

The finding that SGP28 mRNA is so highly expressed in prostate cancers,and not in most normal tissue, provides evidence that this gene isassociated with dysregulated cell growth and therefore identifies thisgene and its products as targets that the skilled artisan can use toevaluate biological samples from individuals suspected of having adisease associated with SGP28 dysregulation. In another example, becausethe expression of SGP28 is normally restricted to prostate and ovary,one can also evaluate biological samples taken from other tissues todetect SGP28 expression as an indication of metastasis. In this context,the evaluation of the expression status of SGP28 gene and its productscan be used to gain information on the disease potential of a tissuesample. The terms “expression status” in this context is used to broadlyrefer to the variety of factors involved in the expression, function andregulation of a gene and its products such as the level of mRNAexpression, the integrity of the expressed gene products (such as thenucleic and amino acid sequences) and transcriptional and translationalmodifications to these molecules.

The expression status of SGP28 may provide information useful forpredicting susceptibility to particular disease stages, progression,and/or tumor aggressiveness. The invention provides methods and assaysfor determining SGP28 expression status and diagnosing cancers thatexpress SGP28, such as cancers of the prostate. SGP28 expression statusin patient samples may be analyzed by a number of means well known inthe art, including without limitation, immunohistochemical analysis, insitu hybridization, RT-PCR analysis on laser capture micro-dissectedsamples, western blot analysis of clinical samples and cell lines, andtissue array analysis. Typical protocols for evaluating the expressionstatus of the SGP28 gene and gene products can be found, for example inCurrent Protocols In Molecular Biology, Units 2 [Northern Blotting], 4[Southern Blotting], 15 [Immunoblotting] and 18 [PCR Analysis],Frederick M. Ausubul et al. eds., 1995.

In one aspect, the invention provides methods for monitoring SGP28 geneproducts by determining the status of SGP28 gene products expressed bycells in a test tissue sample from an individual suspected of having adisease associated with dysregulated cell growth (such as hyperplasia orcancer) and then comparing the status so determined to the status ofSGP28 gene products in a corresponding normal sample, the presence ofaberrant or altered status of SGP28 gene products in the test samplerelative to the normal sample providing an indication of the presence ofdysregulated cell growth within the cells of the individual.

The invention additionally provides methods of examining a biologicalsample for evidence of dysregulated cellular growth. In one embodiment,the method comprises comparing the status of SGP28 in the biologicalsample to the status of SGP28 in a corresponding normal sample, whereinalterations in the status of SGP28 in the biological sample areassociated with dysregulated cellular growth. The status of SGP28 in thebiological sample can be evaluated by, for example, examining levels ofSGP28 mRNA expression or levels of SGP28 protein expression. In oneembodiment, an alteration in the status of SGP28 is identified by thepresence of SGP28 expressing cells in a biological sample from a tissuein which SGP28 expressing cells are normally absent.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in SGP28 mRNA or protein expression in a test cellor tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of SGP28 mRNA may, for example, beevaluated in tissue samples including but not limited to colon, lung,prostate, pancreas, bladder, breast, ovary, cervix, testis, head andneck, brain, stomach, bone, etc. The presence of significant SGP28expression in any of these tissues may be useful to indicate theemergence, presence and/or severity of these cancers or a metastasis ofcancer originating in another tissue, since the corresponding normaltissues do not express SGP28 mRNA or express it at lower levels.

In a related embodiment, SGP28 expression status may be determined atthe protein level rather than at the nucleic acid level. For example,such a method or assay would comprise determining the level of SGP28protein expressed by cells in a test tissue sample and comparing thelevel so determined to the level of SGP28 expressed in a correspondingnormal sample. In one embodiment, the presence of SGP28 protein isevaluated, for example, using immunohistochemical methods. SGP28antibodies or binding partners capable of detecting SGP28 proteinexpression may be used in a variety of assay formats well known in theart for this purpose.

In other related embodiments, one can evaluate the integrity SGP28nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like. Such embodiments areuseful because perturbations in the nucleotide and amino acid sequencesare observed in a large number of proteins associated with a growthdysregulated phenotype (see e.g. Marrogi et al., J. Cutan. Pathol.26(8): 369-378 (1999)). In this context, a wide variety of assays forobserving perturbations in nucleotide and amino acid sequences are wellknown in the art. For example, the size and structure of nucleic acid oramino acid sequences of SGP28 gene products may be observed by thenorthern, Southern, western, PCR and DNA sequencing protocols discussedherein. In addition, other methods for observing perturbations innucleotide and amino acid sequences such as single strand conformationpolymorphism analysis are well known in the art (see e.g. U.S. Pat. Nos.5,382,510 and 5,952,1.70).

In another embodiment, one can examine the methylation status of theSGP28 gene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in >90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas (De Marzoet al., 1999, Am. J. Pathol. 155(6): 1985-1992). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al., 1998, Cancer Epidemiol.Biomarkers Prev., 7:531-536).

In another example, expression of the LAGE-I tumor specific gene (whichis not expressed in normal prostate but is expressed in 25-50% ofprostate cancers) is induced by deoxy-azacytidine in lymphoblastoidcells, suggesting that tumoral expression is due to demethylation (Letheet al., 1998, Int. J. Cancer 76(6): 903-908). In this context, a varietyof assays for examining methylation status of a gene are well known inthe art. For example, one can utilize in Southern hybridizationapproaches methylation-sensitive restriction enzymes which can notcleave sequences that contain methylated CpG sites in order to assessthe overall methylation status of CpG islands.

In addition, MSP (methylation specific PCR) can rapidly profile themethylation status of all the CpG sites present in a CpG island of agiven gene. This procedure involves initial modification of DNA bysodium bisulfite (which will convert all unmethylated cytosines touracil) followed by amplification using primers specific for methylatedversus unmethylated DNA. Protocols involving methylation interferencecan also be found for example in Current Protocols In Molecular Biology,Units 12, Frederick M. Ausubel et al. eds., 1995.

In another related embodiment, the invention provides assays useful indetermining the presence of cancer in an individual, comprisingdetecting a significant change in the SGP28 alternative splice variantsexpressed in a test cell or tissue sample relative to expression levelsin the corresponding normal cell or tissue. The monitoring ofalternative splice variants of SGP28 is useful because changes in thealternative splicing of proteins is suggested as one of the steps in aseries of events that lead to the progression of cancers (see e.g.Carstens et al., Oncogene 15(250: 3059-3065 (1997)).

Gene amplification provides an additional method of assessing the statusof SGP28. Gene amplification may be measured in a sample directly, forexample, by conventional Southern blotting, northern blotting toquantitate the transcription of mRNA Thomas, Proc. Natl. Acad. Sci. USA,77:5201-5205 (1980)], dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

In addition to the tissues discussed above, peripheral blood may beconveniently assayed for the presence of cancer cells, including but notlimited to prostate cancers, using RT-PCR to detect SGP28 expression.The presence of RT-PCR amplifiable SGP28 mRNA provides an indication ofthe presence of the cancer. RT-PCR detection assays for tumor cells inperipheral blood are currently being evaluated for use in the diagnosisand management of a number of human solid tumors. In the prostate cancerfield, these include RT-PCR assays for the detection of cells expressingPSA and PSM (Verkaik et al., 1997, Urol. Res. 25: 373-384; Ghossein etal., 1995, J. Clin. Oncol. 13: 1195-2000; Heston et al., 1995, Clin.Chem. 41: 1687-1688). RT-PCR assays are well known in the art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detectingSGP28 mRNA or SGP28 protein in a tissue sample, its presence indicatingsusceptibility to cancer, wherein the degree of SGP28 mRNA expressionpresent is proportional to the degree of susceptibility. In a specificembodiment, the presence of SGP28 in prostate tissue is examined, withthe presence of SGP28 in the sample providing an indication of prostatecancer susceptibility (or the emergence or existence of a prostatetumor). In a closely related embodiment, one can evaluate the integritySGP28 nucleotide and amino acid sequences in a biological sample inorder to identify perturbations in the structure of these molecules suchas insertions, deletions, substitutions and the like, with the presenceof one or more perturbations in SGP28 gene products in the sampleproviding an indication of cancer susceptibility (or the emergence orexistence of a tumor).

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of SGP28 mRNAor SGP28 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of SGP28 mRNA or SGP28 proteinexpressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree ofSGP28 mRNA or SGP28 protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness. In a specificembodiment, aggressiveness of prostate tumors is evaluated bydetermining the extent to which SGP28 is expressed in the tumor cells,with higher expression levels indicating more aggressive tumors. In aclosely related embodiment, one can evaluate the integrity SGP28nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, with the presence ofone or more perturbations indicating more aggressive tumors.

Yet another related aspect of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of SGP28 mRNA orSGP28 protein expressed by cells in a sample of the tumor, comparing thelevel so determined to the level of SGP28 mRNA or SGP28 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of SGP28 mRNA or SGP28 proteinexpression in the tumor sample over time provides information on theprogression of the cancer. In a specific embodiment, the progression ofa cancer is evaluated by determining the extent to which SGP28expression in the tumor cells alters over time, with higher expressionlevels indicating a progression of the cancer. In a closely relatedembodiment, one can evaluate the integrity SGP28 nucleotide and aminoacid sequences in a biological sample in order to identify perturbationsin the structure of these molecules such as insertions, deletions,substitutions and the like, with the presence of one or moreperturbations indicating a progression of the cancer.

The above diagnostic approaches may be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof SGP28 gene and SGP28 gene products (or perturbations in SGP28 geneand SGP28 gene products) and a factor that is associated with malignancyas a means of diagnosing and prognosticating the status of a tissuesample. In this context, a wide variety of factors associated withmalignancy may be utilized such as the expression of genes otherwiseassociated with malignancy (including PSA, PSCA and PSM expression) aswell as gross cytological observations (see e.g. Bocking et al., 1984,Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. February1995;26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51;Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods forobserving a coincidence between the expression of SGP28 gene and SGP28gene products (or perturbations in SGP28 gene and SGP28 gene products)and an additional factor that is associated with malignancy are useful,for example, because the presence of a set or constellation of specificfactors that coincide provides information crucial for diagnosing andprognosticating the status of a tissue sample.

In a typical embodiment, methods for observing a coincidence between theexpression of SGP28 gene and SGP28 gene products (or perturbations inSGP28 gene and SGP28 gene products) and a factor that is associated withmalignancy entails detecting the overexpression of SGP28 mRNA or proteinin a tissue sample, detecting the overexpression of PSA mRNA or proteinin a tissue sample, and observing a coincidence of SGP28 mRNA or proteinand PSA mRNA or protein overexpression. In a specific embodiment, theexpression of SGP28 and PSA mRNA in prostate tissue is examined. In apreferred embodiment, the coincidence of SGP28 and PSA mRNAoverexpression in the sample provides an indication of prostate cancer,prostate cancer susceptibility or the emergence or existence of aprostate tumor.

Methods for detecting and quantifying the expression of SGP28 mRNA orprotein are described herein and use standard nucleic acid and proteindetection and quantification technologies well known in the art.Standard methods for the detection and quantification of SGP28 mRNAinclude in situ hybridization using labeled SGP28 riboprobes, northernblot and related techniques using SGP28 polynucleotide probes, RT-PCRanalysis using primers specific for SGP28, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like. In a specific embodiment, semi-quantitative RT-PCR may be usedto detect and quantify SGP28 mRNA expression as described in theExamples that follow. Any number of primers capable of amplifying SGP28may be used for this purpose, including but not limited to the variousprimer sets specifically described herein. Standard methods for thedetection and quantification of protein may be used for this purpose. Ina specific embodiment, polyclonal or monoclonal antibodies specificallyreactive with the wild-type SGP28 protein may be used in animmunohistochemical assay of biopsied tissue. Antibodies directedagainst SGP28 protein can also be used to detect SGP28 in a patientspecimen (e.g., blood, urine, semen or other sample) using conventionaltechniques such as fluorescence-activated cell sorting (FACS) and/orELISA.

Identifying Molecules that Interact with SGP28

The SGP28 protein sequences disclosed herein allow the skilled artisanto identify proteins, small molecules and other agents that interactwith SGP28 and pathways activated by SGP28 via any one of a variety ofart accepted protocols. For example one can utilize one of the varietyof so-called interaction trap systems (also referred to as the“two-hybrid assay”). In such systems, molecules that interactreconstitute a transcription factor and direct expression of a reportergene, the expression of which is then assayed. Typical systems identifyprotein-protein interactions in vivo through reconstitution of aeukaryotic transcriptional activator and are disclosed for example inU.S. Pat. Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.

Alternatively one can identify molecules that interact with SGP28protein sequences by screening peptide libraries. In such methods,peptides that bind to selected receptor molecules such as SGP28 areidentified by screening libraries that encode a random or controlledcollection of amino acids. Peptides encoded by the libraries areexpressed as fusion proteins of bacteriophage coat proteins, andbacteriophage particles are then screened against the receptors ofinterest. Peptides having a wide variety of uses, such as therapeutic ordiagnostic reagents, may thus be identified without any priorinformation on the structure of the expected ligand or receptormolecule. Typical peptide libraries and screening methods that can beused to identify molecules that interact with SGP28 protein sequencesare disclosed for example in U.S. Pat. Nos. 5,723,286 and 5,733,731.

Alternatively, cell lines expressing SGP28 can be used to identifyprotein-protein interactions mediated by SGP28. This possibility can beexamined using immunoprecipitation techniques as shown by others(Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).Typically SGP28 protein can be immunoprecipitated from SGP28 expressingprostate cancer cell lines using anti-SGP28 antibodies. Alternatively,antibodies against His-tag can be used in a cell line engineered toexpress SGP28 (vectors mentioned above). The immunoprecipitated complexcan be examined for protein association by procedures such as westernblotting, ³⁵S-methionine labeling of proteins, protein microsequencing,silver staining and two dimensional gel electrophoresis.

Small molecules that interact with SGP28 can be identified throughrelated embodiments of such screening assays. For example, smallmolecules can be identified that interfere with SGP28 function,including molecules that interfere with SGP28's ability to bind to cellsand/or to modulate tumor formation, progression, migration and/orapoptosis. Typical methods are discussed for example in U.S. Pat. No.5,928,868 and include methods for forming hybrid ligands in which atleast one ligand is a small molecule. In an illustrative embodiment, thehybrid ligand is introduced into cells that in turn contain a first anda second expression vector. Each expression vector includes DNA forexpressing a hybrid protein that encodes a target protein linked to acoding sequence for a transcriptional module. The cells further containsa reporter gene, the expression of which is conditioned on the proximityof the first and second hybrid proteins to each other, an event thatoccurs only if the hybrid ligand binds to target sites on both hybridproteins. Those cells that express the reporter gene are selected andthe unknown small molecule or the unknown hybrid protein is identified.

A typical embodiment of this invention consists of a method of screeningfor a molecule that interacts with a SGP28 amino acid sequence shown inTable 2 (SEQ ID NO: 3), comprising the steps of contacting a populationof molecules with the SGP28 amino acid sequence, allowing the populationof molecules and the SGP28 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the SGP28 amino acid sequence and thenseparating molecules that do not interact with the SGP28 amino acidsequence from molecules that do interact with the SGP28 amino acidsequence. In a specific embodiment, the method further includespurifying a molecule that interacts with the SGP28 amino acid sequence.In a preferred embodiment, the SGP28 amino acid sequence is contactedwith a library of peptides. Additional assays for identifying moleculesthat modulate SGP28 function are described in the Examples that follow.

Therapeutic Methods and Compositions

The identification of SGP28 as a prostate cancer protein opens a numberof therapeutic approaches to the treatment of prostate cancers. Asdiscussed above, SGP28 is a secreted protein, and its interaction withother cells and molecules likely plays a role in the regulation of theprostate environment and the initiation, development and/or progressionof cancer. SGP28 can be targeted for therapy via approaches aimed atinhibiting activity of the SGP28 protein, inhibiting the binding orassociation of SGP28 protein with other cells and molecules, inhibitingtranscription or translation of SGP28, and/or via the use of cancervaccines based on SGP28. The therapeutic strategy can thus be designedto inhibit a function of the molecule or to target the SGP28 moleculeitself.

The expression profile of SGP28 is reminiscent of the MAGEs, PSA andPMSA, which are tissue-specific genes that are up-regulated in melanomasand other cancers (Van den Eynde and Boon, Int J Clin Lab Res.27:81-86,1997). Due to their tissue-specific expression and highexpression levels in cancer, these molecules are currently beinginvestigated as targets for cancer vaccines (Durrant, Anticancer Drugs8:727-733, 1997; Reynolds et al., Int J Cancer 72:972-976, 1997). Theexpression pattern of SGP28 provides evidence that it is likewise anideal target for a cancer vaccine approach to prostate cancer, as itsexpression is not detected in most normal tissues.

Accordingly, therapeutic approaches targeting particular motifs ofSGP28, or aimed at inhibiting the activity of the SGP28 protein, areexpected to be useful for patients suffering from prostate cancer andother cancers expressing SGP28. The therapeutic approaches aimed atinhibiting the activity of the SGP28 protein generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of the SGP28 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of the SGP28 gene or translation of SGP28mRNA.

SGP28 as a Target for Antibody-Based Therapy

The SGP28 molecule is an attractive target for antibody-basedtherapeutic strategies. Because SGP28 is expressed in cancer cells andnot in most normal tissues, systemic administration ofSGP28-immunoreactive compositions would be expected to exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunotherapeutic molecule to non-target organs andtissues. Antibodies specifically reactive with SGP28 can be useful totreat SGP28-expressing cancers systemically, either as conjugates with atoxin or therapeutic agent, or as naked antibodies capable of inhibitinginteraction of SGP28 with its binding partner.

SGP28 antibodies can be introduced into a patient such that the antibodybinds to SGP28 and eliminates SGP28 function in the primary tumor, incirculating micrometastases, and/or in established metastases. Thedegree of tumor vascularization may provide guidance on which deliveryapproach is recommended. Similary, the grade and/or stage of diseasewould be expected to provide useful information in this regard. Forexample, a higher grade, more advanced tumor may be more likely to seedmetastases, suggesting systemic administration in order to treat orprevent the emergence of metastases.

Cancer immunotherapy using anti-SGP28 antibodies may follow theteachings generated from various approaches that have been successfullyemployed in the treatment of other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol.18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186;Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk etal., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al.,1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhonget al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,1994, Cancer Res. 54:6160-6166); Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20antibodies (e.g., Bexxar, Coulter Pharmaceutical), while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Fortreatment of prostate cancer, for example, SGP28 antibodies can beadministered in conjunction with radiation, chemotherapy or hormoneablation.

Although SGP28 antibody therapy may be useful for all stages of cancer,antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionmay be indicated for patients who have received previously one or morechemotherapy, while combining the antibody therapy of the invention witha chemotherapeutic or radiation regimen may be preferred for patientswho have not received chemotherapeutic treatment. Additionally, antibodytherapy may enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

It may be desirable for some cancer patients to be evaluated for thepresence and level of SGP28 expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative SGP28imaging, or other techniques capable of reliably indicating the presenceand degree of SGP28 expression. Immunohistochemical analysis of tumorbiopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

Anti-SGP28 monoclonal antibodies useful in treating prostate and othercancers include those that are capable of initiating a potent immuneresponse against the tumor and those that are capable of directcytotoxicity. In this regard, anti-SGP28 monoclonal antibodies (mAbs)may elicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, anti-SGP28 mAbs that exert a direct biological effect ontumor growth are useful in the practice of the invention. Potentialmechanisms by which such directly cytotoxic mAbs may act includeinhibition of cell growth, modulation of cellular differentiation,modulation of tumor angiogenesis factor profiles, and the induction ofapoptosis. The mechanism by which a particular anti-SGP28 mAb exerts ananti-tumor effect may be evaluated using any number of in vitro assaysdesigned to determine ADCC, ADMMC, complement-mediated cell lysis, andso forth, as is generally known in the art.

The use of mutine or other non-human monoclonal antibodies, orhuman/mouse chimeric mAbs may induce moderate to strong immune responsesin some patients. In some cases, this will result in clearance of theantibody from circulation and reduced efficacy. In the most severecases, such an immune response may lead to the extensive formation ofimmune complexes that, potentially, can cause renal failure.Accordingly, preferred monoclonal antibodies used in the practice of thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target SGP28antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-SGP28 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails may have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination mayexhibit synergistic therapeutic effects. In addition, the administrationof anti-SGP28 mAbs may be combined with other therapeutic agents,including but not limited to various chemotherapeutic agents,androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF). Theanti-SGP28 mabs may be administered in their “naked” or unconjugatedform, or may have therapeutic agents conjugated to them.

The anti-SGP28 antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumor, intradermal,and the like. Treatment will generally involve the repeatedadministration of the anti-SGP28 antibody preparation via an acceptableroute of administration such as intravenous injection (IV), typically ata dose in the range of about 0.1 to about 10 mg/kg body weight. Doses inthe range of 10-500 mg mAb per week may be effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-SGP28 mAb preparation may represent an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the Ab or mAbs used, the degree of SGP28expression in the patient, the extent of circulating shed SGP28 antigen,the desired steady-state antibody concentration level, frequency oftreatment, and the influence of chemotherapeutic agents used incombination with the treatment method of the invention.

Optimally, patients should be evaluated for the level of circulatingshed SGP28 antigen in serum in order to assist in the determination ofthe most effective dosing regimen and related factors. Such evaluationsmay also be used for monitoring purposes throughout therapy, and may beuseful to gauge therapeutic success in combination with evaluating otherparameters (such as serum PSA levels in prostate cancer therapy).

Inhibition of SGP28 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of SGP28 to its binding partner or ligand, or itsassociation with other protein(s) as well as methods for inhibitingSGP28 function.

Inhibition of SGP28 With Recombinant Proteins

In one approach, recombinant molecules that are capable of binding toSGP28 thereby preventing SGP28 from accessing/binding to its bindingpartner(s) or associating with other protein(s) are used to inhibitSGP28 function. Such recombinant molecules may, for example, contain thereactive part(s) of a SGP28 specific antibody molecule. In a particularembodiment, the SGP28 binding domain of a SGP28 binding partner may beengineered into a dimeric fusion protein comprising two SGP28 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion may contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins may be administered in soluble form to patientssuffering from a cancer associated with the expression of SGP28,including but not limited to prostate cancer, where the dimeric fusionprotein specifically binds to SGP28 thereby blocking SGP28 interactionwith a binding partner. Such dimeric fusion proteins may be furthercombined into multimeric proteins using known antibody linkingtechnologies.

Inhibition of SGP28 with Intracellular Antibodies

In another approach, recombinant vectors encoding single chainantibodies that specifically bind to SGP28 may be introduced into SGP28expressing cells via gene transfer technologies, wherein the encodedsingle chain anti-SGP28 antibody is expressed intracellularly, binds toSGP28 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, may bespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatmentwill be focused. This technology has been successfully applied in theart (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors. See, for example, Richardsonet al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies may beexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals maybe engineered into recombinant polynucleotide vectors encoding suchsingle chain antibodies in order to precisely target the expressedintrabody to the desired intracellular compartment. For example,intrabodies targeted to the endoplasmic reticulum (ER) may be engineeredto incorporate a leader peptide and, optionally, a C-terminal ERretention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus may be engineered to include anuclear localization signal. Lipid moieties may be joined to intrabodiesin order to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

In one embodiment, SGP28 intrabodies are designed to bind specificallyto a particular SGP28 domain. For example, cytosolic intrabodies thatspecifically bind to the SGP28 protein may be used to prevent SGP28related molecules from gaining access to the nucleus, thereby preventingit from exerting any biological activity within the nucleus.

In order to direct the expression of such intrabodies specifically toparticular tumor cells, the transcription of the intrabody may be placedunder the regulatory control of an appropriate tumor-specific promoterand/or enhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer may beutilized (See, for example, U.S. Pat. No. 5,919,652).

Inhibition of SGP28 Transcription or Translation

Within another class of therapeutic approaches, the invention providesvarious methods and compositions for inhibiting the transcription of theSGP28 gene. Similarly, the invention also provides methods andcompositions for inhibiting the translation of SGP28 mRNA into protein.

In one approach, a method of inhibiting the transcription of the SGP28gene comprises contacting the SGP28 gene with a SGP28 antisensepolynucleotide. In another approach, a method of inhibiting SGP28 mRNAtranslation comprises contacting the SGP28 mRNA with an antisensepolynucleotide. In another approach, a SGP28 specific ribozyme may beused to cleave the SGP28 message, thereby inhibiting translation. Suchantisense and ribozyme based methods may also be directed to theregulatory regions of the SGP28 gene, such as the SGP28 promoter and/orenhancer elements. Similarly, proteins capable of inhibiting a SGP28gene transcription factor may be used to inhibit SGP28 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of SGP28 throughinterfering with SGP28 transcriptional activation may also be useful forthe treatment of cancers expressing SGP28. Similarly, factors that arecapable of interfering with SGP28 processing may be useful for thetreatment of cancers expressing SGP28. Cancer treatment methodsutilizing such factors are also within the scope of the invention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies may be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizing SGP28(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother SGP28 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding SGP28 antisensepolynucleotides, ribozymes, factors capable of interfering with SGP28transcription, and so forth, may be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches may be combined with any one of a widevariety of chemotherapy or radiation therapy regimens. These therapeuticapproaches may also enable the use of reduced dosages of chemotherapyand/or less frequent administration, particularly in patients that donot tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, may beevaluated using various in vitro and in vivo assay systems. In vitroassays for evaluating therapeutic potential include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of SGP28 to a bindingpartner, etc.

In vivo, the effect of a SGP28 therapeutic composition may be evaluatedin a suitable animal model. For example, xenogeneic prostate cancermodels wherein human prostate cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice, are appropriate in relation to prostate cancer and have beendescribed (Klein et al., 1997, Nature Medicine 3: 402-408). For example,PCT Patent Application WO98/16628, Sawyers et al., published Apr. 23,1998, describes various xenograft models of human prostate cancercapable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

In vivo assays that qualify the promotion of apoptosis may also beuseful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions, includingvaccine compositions, comprising a carrier suitable for the desireddelivery method. Suitable carriers include any material that whencombined with the therapeutic composition retains the anti-tumorfunction of the therapeutic composition and is non-reactive with thepatient's immune system. Examples include, but are not limited to, anyof a number of standard pharmaceutical carriers such as sterilephosphate buffered saline solutions, bacteriostatic water, and the like(see, generally, Remington's Pharmaceutical Sciences 16^(th) Edition, A.Osal., Ed., 1980).

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations may be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancerand will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

The invention further provides cancer vaccines comprising a SGP28protein or fragment thereof, as well as DNA based vaccines. In view ofthe prostate- and tumor-restricted expression of SGP28, SGP28 cancervaccines are expected to be effective at specifically preventing and/ortreating SGP28 expressing cancers without creating non-specific effectson non-target tissues. The use of a tumor antigen in a vaccine forgenerating humoral and cell-mediated immunity for use in anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995,Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159:3113-3117). Such methods can be readily practiced by employing a SGP28protein, or fragment thereof, or a SGP28-encoding nucleic acid moleculeand recombinant vectors capable of expressing and appropriatelypresenting the SGP28 immunogen.

For example, viral gene delivery systems may be used to deliver aSGP28-encoding nucleic acid molecule. Various viral gene deliverysystems that can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a SGP28 protein or fragment thereof introduced into the patient(e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the fill-length human SGP28 cDNA may be employed.

In one embodiment, a SGP28 cancer vaccine is based on the identificationof immunogenic peptides within the SGP28 amino acid sequence shown inTable 2 (SEQ ID NO: 3). As discussed further in the examples below,SGP28 has been shown to induce T and B cell responses. A recombinantHIS-tagged protein including the ORF of SGP28 (Table 2; SEQ ID NO: 3)has been used to generate an immune response in mice for the productionof monoclonal antibodies. Amino acids 93-107 of SGP28 (CNYRHSNPKDRMTSL;SEQ ID NO: 27), have been used to generate an immune response in rabbitsfor the production of polyclonal antibodies. Thus, specific portions ofSGP28, and polynucleotides encoding these portions, may be selected forthe production of a cancer vaccine.

In another embodiment, SGP28 nucleic acid molecules encoding specificcytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes canbe determined using specific algorithms (e.g., Epimer, Brown University)to identify peptides within a SGP28 protein that are capable ofoptimally binding to specified HLA alleles. One suitable algorithm isthe HLA Peptide Motif Search algorithm available at the Bioinformaticsand Molecular Analysis Section (BIMAS) web site(http://bimas.dcrt.nih.gov/). This algorithm is based on binding ofspecific peptide sequences in the groove of HLA Class I molecules andspecifically HLA-A2 (Falk et al., 1991, Nature 351:290-6; Hunt et al.,1992, Science 255:1261-3; Parker et al., 1992, J. Immunol. 149:3580-7;Parker et al., 1994, J. Immunol. 152:163-75). The HLA Peptide MotifSearch algorithm allows location and ranking of 8mer, 9-mer, and 10-merpeptides from a complete protein sequence for predicted binding toHLA-A2 as well as other Class I molecules. Most HLA-A2 binding peptidesare 9-mers, favorably containing a leucine at position 2 and a valine orleucine at position 9 (Parker et al., 1992, J. Immunol. 149:3580-7).Actual binding of peptides to HLA-A2 can be evaluated by stabilizationof HLA-A2 expression on the antigen processing defective cell line T2(Xue et al., 1997, Prostate 30:73-8; Peshwa et al., 1998, Prostate36:129-38). Immunogenicity of specific peptides can be evaluated invitro by stimulation of CD8+ CTL in the presence of dendritic cells (Xueet al.; Peshwa et al., supra).

Specific SGP28 peptides predicted to bind HLA-A2 and preferred for usein cancer vaccines include peptides corresponding to amino acids 2-10(TLFPVLLFL; SEQ ID NO: 17), amino acids 6-14 (VLLFLVAGL; SEQ ID NO: 18),amino acids 30-38 (ALLTTQTQV; SEQ ID NO: 19), amino acids 142-150(VVWYSSYLV; SEQ ID NO: 20), amino acids 222-230 (TLTCKHQLV; SEQ ID NO:21), amino acids 175-183 (GNWANRLYV; SEQ ID NO: 22), amino acids 7-15(LLFLVAGLL; SEQ ID NO: 23), amino acids 141-149 (QVVWYSSYL; SEQ ID NO:24), amino acids 134-142 (AVVGHYTQV; SEQ ID NO: 25), and amino acids211-219 (DLYSNCKSL; SEQ ID NO: 26) of the SGP28 protein sequence shownin Table 2.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present SGP28 antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al.,1996, Prostate 29: 371-380). Dendritic cells can be used to presentSGP28 peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed withSGP28 peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete SGP28 protein.Yet another embodiment involves engineering the overexpression of theSGP28 gene in dendritic cells using various implementing vectors knownin the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther.4: 17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribaset al., 1997, Cancer Res. 57: 2865-2869), and tumor-derived RNAtransfection (Ashley et al., 1997, J. Exp. Med. 186: 1177-1182). Cellsexpressing SGP28 may also be engineered to express immune modulators,such as GM-CSF, and used as immunizing agents.

Anti-idiotypic anti-SGP28 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga SGP28 protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-SGP28 antibodies that mimic an epitope on aSGP28 protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al.,1996, Cancer Immunol Immunother 43: 65-76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing SGP28. Constructs comprising DNA encoding aSGP28 protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take up the construct and express theencoded SGP28 protein/immunogen. Expression of the SGP28 proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against prostate and otherSGP28-expressing cancers. Various prophylactic and therapeutic geneticimmunization techniques known in the art may be used (for review, seeinformation and references published at Internet addresswww.genweb.com).

Diagnostic Compositions and Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a SGP28 proteinor a SGP28 gene or message, respectively. Where the kit utilizes nucleicacid hybridization to detect the target nucleic acid, the kit may alsohave containers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

Accordingly, the invention also provides diagnostic compositionscomprising SGP28-related molecules. Such molecules include the variousSGP28 polynucleotides, primers, probes, proteins, fragments, antibodiesdescribed herein. The molecules included in the diagnostic compositionmay optionally be labeled with a detectable marker. SGP28 diagnosticcompositions may further comprise appropriate buffers, diluents, andother ingredients as desired.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the SGP28 Gene

Materials and Methods

LAPC Xenografts:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craftet al., 1999, Cancer Res. 59: 5030-5036). Androgen dependent andindependent LAPC-4 xenografts (LAPC-4 AD and Al, respectively) andLAPC-9 xenografts (LAPC-9 AD and Al, respectively) were grown in intactmale SCID mice or in castrated males, respectively, and were passaged assmall tissue chunks in recipient males. LAPC-4 AI xenografts werederived from LAPC-4 AD tumors and LAPC-9 AI xenografts were derived fromLAPC-9 AD tumors. To generate the AI xenografts, male mice bearing LAPCAD tumors were castrated and maintained for 2-3 months. After the LAPCtumors re-grew, the tumors were harvested and passaged in castratedmales or in female SCID mice.

LAPC-4 AD xenografts were grown intratibially as follows. LAPC-4 ADxenograft tumor tissue grown subcutaneously was minced into 1-2 mm³sections while the tissue was bathed in 1× Iscoves medium, minced tissuewas then centrifuged at 1.3 K rpm for 4 minutes, the supernatant wasresuspended in 10 ml ice cold 1× Iscoves medium and centrifuged at 1.3 Krpm for 4 minutes. The pellet was then resuspended in 1× Iscoves with 1%pronase E and incubated for 20 minutes at room temperature with mildrocking agitation followed by incubation on ice for 2-4 minutes.Filtrate was centrifuged at 1.3K rmp for 4 minutes, and the pronase wasremoved from the aspirated pellet by resuspending in 10 ml Iscoves andre-centrifuging. Clumps of cells were then plated in PrEGM medium andgrown overnight. The cells were then harvested, filtered, washed 2×RPMI, and counted. Approximately 50,000 cells were mixed with and equalvolume of ice-cold Matrigel on ice, and surgically injected into theproximal tibial metaphyses of SCID mice via a 27 gauge needle. After10-12 weeks, LAPC-4 tumors growing in bone marrow were recovered.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used. DPNCDN (cDNAsynthesis primer): (SEQ ID NO: 28) 5′TTTTGATCAAGCTT₃₀3′ Adaptor 1: (SEQID NO: 29,30) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 31,32)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ 3′CGGCTCCTAG5′ PCR primer1: (SEQ ID NO: 33) 5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP)1: (SEQID NO: 34) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP)2: (SEQ ID NO:35) 5′AGCGTGGTCGCGGCCGAGGA3′Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes which may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from LAPC-4 AD xenografts growingin two different environments, namely the subcutaneous (“LAPC-4 AD SQ”)and intratibial (“LAPC-4 AD IT”) growth environments, wherein the LAPC-4AD IT xenograft was used as the source of the “tester” cDNA, while theLAPC-4 AD SQ xenograft was used as the source of the “driver” cDNA.

Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)+ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs. at 37° C.Digested cDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant xenograft source (see above) with a mix ofdigested cDNAs derived from human benign prostatic hyperplasia (BPH),the human cell lines HeLa, 293, A431, Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant xenograft source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification. Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl DNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dbest, and NCI-CGAP databases.

Results

The SSH experiment described in the Materials and Methods, supra, led tothe isolation of numerous candidate gene fragment clones (SSH clones).All candidate clones were sequenced and subjected to homology analysisagainst all sequences in the major public gene and EST databases inorder to provide information on the identity of the corresponding geneand to help guide the decision to analyze a particular gene fordifferential expression. In general, gene fragments which had nohomology to any known sequence in any of the searched databases, andthus considered to represent novel genes, as well as gene fragmentsshowing homology to previously sequenced expressed sequence tags (ESTs),were subjected to differential expression analysis by RT-PCR and/orNorthern analysis.

One of the SSH clones was identical to the corresponding sequence of asecreted molecule known as specific granule protein 28 (SGP28) (Kjeldsenet al., 1996, FEBS Lett. 380, 246-250) or cysteine-rich secretoryprotein (CRISP-3) (Kratzschmar et al., 1996, Eur J Biochem236(3):827-36). The sequence of this clone (36P1G3) is as follows: (SEQID NO: 1) GATCTCTATAGTAACTGTAAAAGTTTGAAGCTCACATTAACCTGTAAACATCAGTTGGTCAGGGACAGTTGCAAGGCCTCCTGCAATTGTTCAAACAGCATTTATTAAATACGCATTACACACCGAGTAGGGCTATGTAGAGAGGAGTCAGATTATCTACTTAGATTTGGCATCTACTTAGATTTAACATATACTAGCTGAGAAATTGTAGGCATGTTTGATACACATTTGATTTCAAATGTTTTTCTTC TGGATC.

Example 2 Isolation of Full Length SGP28 Encoding cDNA

A full length cDNA (clone 1; Table 1) of 774 bp was isolated from aprostate library, revealing an ORF of 258 amino acids (Table 2). Thesequence identified herein differs from the published SGP28 sequence(Kjeldsen et al., 1996, FEBS Lett. 380(3):246-50) in two nucleic acids,one in the coding sequence and one in the 5′ UTR. These differences donot alter the protein sequence. TABLE 1 Full Length 36P1G3/SGP28 cDNA(SEQ ID NO: 2) TGATGAAACAAATACTTCATCCTGCTCTGGAAACCACTGCAATGACATTATTCCCAGTGCTGTTGTTCCTGGTTGCTGGGCTGCTTCCATCTTTTCCAGCAAATGAAGATAAGGATCCCGCTTTTACTGCTTTGTTAACCACCCAAACACAAGTGCAAAGGGAGATTGTGAATAAGCACAATGAACTGAGGAGAGCAGTATCTCCCCCTGCCAGAAACATGCTGAAGATGGAATGGAACAAAGAGGCTGCAGCAAAATGCCCAAAAGTGGGCAAACCAGTGCAATTACAGACACAGTAACCCAAAGGATCGAATGACAAGTCTAAAATGTGGTGAGAATCTCTACATGTCAAGTGCCCCCAGCTCATGGTCACAAGCAATCCAAAGCTGGTTTGATGAGTACAATGATTTTGACTTTGGTGTAGGGCCAAAGACTCCCAACGCAGTGGTTGGACATTATACACAGGTTGTTTGGTACTCTTCATACCTCGTTGGATGTGGAAATGCCTACTGTCCCAATCAAAAAGTTCTAAAATACTACTATGTTTGCCAATATTGTCCTGCTGGTAATTGGGCTAATAGACTATATGTCCCTTATGAACAAGGAGCACCTTGTGCCAGTTGCCCAGATAACTGTGACGATGGACTATGCACCAATGGTTGCAAGTACGAAGATCTCTATAGTAACTGTAAAAGTTTGAAGCTCACATTAACCTGTAAACATCAGTTGGTCAGGGACAGTTGCAAGGCATCCTGCAATTGTTCAAACAGCATTTATTAAATACGCATTACACACCGAGTAGGGCTATGTAGAGAGGAGTCAGATTATCTACTTAGATTTGGCATCTACTTAGATTTAACATATACTAGCTGAGAAATTGTAGGCATGTTTGATACACATTTGATTTCAAATGTTTTTCTTCTGGATCTGCTTTTTATTTTACAAAAATATTTTTCATACAAATGGTTAAAAAGAAACAAAATCTATAACAACAACTTTGGATTTTTATATATAAACTTTGTGATTTAAATTTACTGAATTTAATTAGGGTGAAAATTTTGAAAGTTGTATTCTCATATGACTAAGTTCACTAAAACCCTGGATTGAAAGTGAAAATTATGTTCCTAGAACAAAATGTACAAAAAGAACAATATAATTTTCACATGAACCCTTGGCTGTAGTTGCCTTTCCTAGCTCCACTCTAAGGCTAAGCATCTTCAAAGACGTTTTCCCATATGCTGTCTTAATTCTTTTCACTCATTCACCCTTCTTCCCAATCATCTGGCTGGCATCCTCACAATTGAGTTGAAGCTGTTCCTCCTAAAACAATCCTGACTTTTATTTTGCCAAAATCAATACAATCCTTTGAATTTTTTATCTGCATAAATTTTACAGTAGAATATGATCAAACCTTCATTTTTAAACCTCTCTTCTCTTTGACAAAACTTCCTTAAAAAAGAATACAAGATAATATAGGTAAATACCCTCCACTCAAGGAGGTAGAACTCAGTCCTCTCCCTTGTGAGTCTTCACTAAAATCAGTGACTCACTTCCAAAGAGTGGAGTATGGAAAGGGAAACATAGTAACTTTACAGGGGAGAAAAATGACAAATGACGTCTTCACCAAGTGATCAAAATTAACGTCACCAGTGATAAGTCATTCAGATTTGTTCTAGATAATCTTTCTAAAAATTCATAATCCCAATCTAATTATGAGCTAAAACATCCAGCAAACTCAAGTTGAAGGACATTCTACAAAATATCCCTGGGGTATTTTAGAGTATTCCTCAAAACTGTAAAAATCATGGAAAATAAGGGAATCCTGAGAAACAATCACAGACCACATGAGACTAAGGAGACATGTGAGCCAAATGCAATGTGCTTCTTGGATCAGATCCTGGAACAGAAAAAGATCAGTAATGAAAAAACTGATGAAGTCTGAATAGAATCTGGAGTATTTTTAACAGTAGTGTTGATTTCTTAATCTTGACAAATATAGCAGGGTAATGTAAGATGATAACGTTAGAGAAACTGAAACTGGGTGAGGGCTATCTAGGAATTCTCTGTACTATCTTACCAAATTTTCGGTAAGTCTAAGAAAGCAATGCAAAATAAAAAGTATCTTGAAAAAAAAAAAAAAAAAAA

TABLE 2 36P1G3/SGP28 Open Reading Frame (SEQ ID NO: 3)MKQILHPALETTAMTLFPVLLFLVAGLLPSFPANEDKDPAFTALLTTQTQVQREIVNKHNELRRAVSPPARNMLKMEWNKEAAANAQKWANQCNYRHSNPKDRMTSLKCGENLYMSSAPSSWSQAIQSWFDEYNDFDFGVGPKTPNAVVGHYTQVVWYSSYLVGCGNAYCPNQKVLKYYYVCQYCPAGNWANRLYVPYEQGAPCASCPDNCDDGLCTNGCKYEDLYSNCKSLKLTLTCKHQLVRDSCKAS CNCSNSIY

Example 3 SGP28 Gene Expression Analysis

SGP28 mRNA expression in normal human tissues was first analyzed bynorthern blotting two multiple tissue blots obtained from Clontech (PaloAlto, Calif.), comprising a total of 16 different normal human tissues,using labeled 36P1G3 cDNA as a probe (sequence as in Example 1). RNAsamples were quantitatively normalized with a β-actin probe. The resultsare shown in FIGS. 1A-B and indicate that, within the 16 tissues tested,the SGP28 gene is exclusively expression in prostate, testis and ovary.Interestingly, the prostate and ovary exhibit a 2.4 kb transcript, whiletestis expresses a 1.6 kb message (the 1.6 kb message could representanother SGP28 family member). The lower molecular weight signal innormal testis is probably due to cross-hybridization of the probe (SSHfragment) to CRISP2 message. An identical transcript is seen for CRISP2on this normal panel using a gene specific oligonucleotide probe in thepublication by Kratzschmar, J. et al., 1996, Eur. J. Biochem.236:827-836.

In addition, in order to analyze SGP28 expression in human cancertissues and cell lines, RNAs derived from LAPC-4 human prostate cancerxenografts were analyzed by northern blot using the 36P1G3 probe. AllRNA samples were quantitatively normalized by ethiduim bromide stainingand subsequent analysis with a labeled β-actin probe. The results ofthis analysis are presented in FIG. 1C, and show very high levelexpression of the 2.4 kb SGP28 transcript in all of the LAPC xenografts.

To analyze SGP28 expression in prostate cancer tissues, northernblotting was performed on RNA derived from three prostate tumor sampleswith their matched normal adjacent prostate tissue. The results showthat SGP28 mRNA expression was detected in all 3 of the 3 tumorspecimens tested, and a very high level of expression in 2 of the 3(FIG. 2).

Example 4 Generation of Polyclonal Antibodies to SGP28

To generate polyclonal sera to SGP28, a peptide was synthesizedcorresponding to amino acids 93-107 (CNYRHSNPKDRMTSL; SEQ ID NO: 27) ofthe SGP28 protein. The peptide sequence was coupled to Keyhole limpethemacyanin (KLH) and was used to immunize a rabbit as follows. Therabbit was initially immunized with 200 μg of peptide-KLH mixed incomplete Freund's adjuvant The rabbit was then injected every two weekswith 200 μg of peptide-KLH in incomplete Freund's adjuvant. Bleeds weretaken approximately 7-10 days following each immunization. ELISA andwestern blotting analyses were used to determine specificity and titerof the rabbit serum to the immunizing peptide and SGP28 proteinrespectively. Affinity purified SGP28 polyclonal antibodies wereprepared by passage of crude serum from immunized rabbit over anaffinity matrix comprised of SGP28 peptide (CNYRHSNPKDRMTSL; SEQ ID NO:27) covalently coupled to Affigel 10 (BioRad). After extensive washingof the matrix with PBS, antibodies specific to SGP28 peptide were elutedwith low pH glycine buffer (0,1 M, pH 2.5), immediately neutralized, anddialyzed extensively against PBS.

To test the rabbit serum for reactivity with SGP28 protein, full lengthSGP28 cDNA was cloned into an expression vector that provides a 6His tagat the carboxyl-terminus (PCDNA 3.1 myc-his, InVitrogen). The resultingMYC/HIS SGP28 construct was transfected into 293T cells. Whole celllysates and supernatants of LAPC4 cells and MYC/HIS SGP28 transientlytransfected 293T cells and LAPC4 and LAPC9 xenograft lysates weresubjected to western blotting using affinity purified rabbit anti-SGP28pAb (1 μg/ml). SGP28 immunoreactive bands were visualized by incubationof the blots with HRP-conjugated anti-rabbit secondary antibody,followed by enhanced chemi-luminescence detection. The results are shownin FIG. 3, and demonstrate that the anti-SGP28 polyclonal antibodyidentifies SGP28 protein in LAPC4 and LAPC9 xenograft lysates and inLAPC4 and transfected 293T cell line supernatants.

Example 5 Expression of Recombinant SGP28 Protein in Mammalian CellspcDNA3.1/MycHis Construct

To express 36P1G3 in mammalian cells, the 774 bp (258 amino acid) 36P1G3ORF was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter. The recombinant protein has the myc and six histidines fusedto the C-terminus. The pcDNA3.1/MycHis vector also contains the bovinegrowth hormone (13GH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells expressing the protein and the ampicillinresistance gene and ColE1 origin permits selection and maintenance ofthe plasmid in E. coli.

pAPtag Construct

The 36P1G3/SGP28 protein without the signal sequence (amino acids 33 to258) was cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). Thisconstruct generates an alkaline phosphatase fusion at the C-terminus ofthe 36P1G3 protein while fusing the IgGK signal sequence to N-terminus.The resulting recombinant 36P1G3 protein is optimized for secretion intothe media of transfected mammalian cells and can be used to identifyproteins such as ligands or receptors that interact with the 36P1G3protein. Protein expression is driven from the CMV promoter and therecombinant protein also contains myc and six histidines fused to theC-terminus of alkaline phosphatase. The Zeosin resistance gene allowsfor selection of mammalian cells expressing the protein and theampicillin resistance gene permits selection of the plasmid in E. coli.

ptag5 Construct

The 36P1G3 protein without the signal sequence (amino acids 33 to 258)was also cloned into pTag-5. This vector is similar to pAPTag butwithout the alkaline phosphatase fusion.

pSRa Constructs

To generate mammalian cell lines expressing 36P1G3 constitutively, the774 bp (258 amino acid) ORF was cloned into pSRa constructs. Amphotropicand ecotropic retroviruses are generated by transfection of pSRaconstructs into the 293T-10A1 packaging line or co-transfection of pSRaand a helper plasmid (φ-) in 293 cells, respectively. The retrovius canbe used to infect a variety of mammalian cell lines, resulting in theintegration of the cloned gene, 36P1G3, into the host cell-lines.Protein expression is driven from a long terminal repeat (LTR). Theneomycin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli. Anadditional pSRa construct was made that fused the FLAG tag to theC-terminus to allow detection using anti-FLAG antibodies. The FLAGsequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 36) wereadded to cloning primer at the 3′ end of the ORF.

Additional pSRa constructs can be made to produce both N-terminal andC-terminal GFP and myc/6 HIS fusion proteins of the full length 36P1G3protein.

Example 6 Expression of Recombinant SGP28 Protein in Insect CellspMelBac a

The 36P1G3 protein without the signal sequence (amino acids 33 to 258)was also cloned into pMelBac (Cat no. V1950-20, Invitrogen, CA) toexpress and secrete the protein in Sf9 insect cells. The pMelBAC Avector is a baculovirus transfer vector designed to direct expression ofrecombinant proteins through the secretory pathway to the extracellularmedium. The signal sequence for honeybee melittin, a high expressed andefficiently secreted protein, is used to direct secretion of therecombinant 36P1G3 protein. Protein expression is driven under thepolyhedrin promoter. A C-terminal myc-his tagged construct was also madein pMelBac A to allow for detection and purification of the recombinant36P1G3 protein.

pIZT/V5His

The 36P1G3 protein was cloned into pIZT/V5His (Cat no. v8010-01,Invitrogen, CA) to express the protein in Sf9 insect cells. Theexpression vector allows for transient and stable expression of therecombinant protein. Protein expression is driven by the OpIE2 promoterfor high-level, constitutive expression. The Zeocin resistance gene isunder the control of the OpIE1 promoter.

Example 7 Production of Monoclonal Antibodies to SGP28

To generate mAbs to SGP28, recombinant HIS-tagged SGP28 protein purifiedfrom 293T tissue culture supernatants was used to immunize 5 female BalbC mice. Initial immunization was carried out with 50 μg of purifiedSGP28 protein mixed in Freund's complete adjuvant. Boosts were thenadministered in 2 week intervals with 50 μg of SGP28 protein mixed inFreund's incomplete adjuvant. Reactivity and specificity of test bleedstaken 7-10 days following each immunization was determined by ELISA andwestern blotting procedures. The specific titer of test bleeds to theimmunogen was at least 2×10⁶. Three mice were subsequently sacrificedand spleens were used to carry out fusion and hybridoma generation usingstandard procedures (Harlow and Lane, 1988). Eleven positive wells werethen subjected to subcloning to generate SGP28-specific monoclonalhybridomas. One of the hybridomas that has completed subcloning,designated 4G6 (IgG1 isotype), specifically recognizes SGP28 proteinpresent in prostate cancer cell lysates and supernatants and markedlyreacts with SGP28 protein in clinical prostate cancer tissue, but not innormal adjacent tissue from the same patient (FIG. 4).

FIG. 4 shows that anti-SGP28 monoclonal antibody specifically detectsSGP28 protein in prostate cancer cell lines and supernatants, prostatecancer xenografts, and clinical prostate cancer tissue. Cell lysate andconditioned media from the LAPC4 prostate cancer cell line and lysatesfrom LAPC4 and LAPC9 prostate cancer xenografts and from a matchednormal and cancerous prostate clinical specimen were separated bySDS-PAGE and transferred to nitrocellulose. The blot was then subjectedto western analysis with a 1:2 dilution of 4G6 anti-SGP28 monoclonalantibody supernatant. Specific SGP28 immunoreactive bands were thenvisualized by incubation with anti-mouse IgG-HRP conjugate secondaryantibody and development with enhanced chemiluminescence and exposure toautoradiographic film. Indicated with arrows is the SGP28 immunoreactiveprotein doublet.

Example 9 Western Analysis of SGP28 Protein Expression

Matched clinical tissue lysates of prostate cancer and normal adjacenttissue, as well as a normal tissue lysates of an LAPC4 cell line andLAPC-4 xenograft were subjected to western blotting with 1 μg/ml ofaffinity purified rabbit anti-SGP28 polyclonal antisera. SGP28immunoreactive bands were visualized by incubation of the blots withHRP-conjugated anti-rabbit secondary antibody followed by enhancedchemiluminescence detection. The results (FIGS. 5A-B) show high levelexpression of SGP28 in the prostate cancer samples but not the adjacentnormal tissue and high level expression in the LAPC xenografts. Lowlevel expression was detected in normal testis and lung.

Example 10 Immunohistochemical Detection of SGP28 in Prostate Cancer,PIN and Prostate Cancer Metastases

SGP28 expression in a Gleason score 7 prostate cancer specimen as wellas a high grade PIN specimen were subjected to immunohistochemicalanalysis of SGP28 expression as follows. Tissue sections were preparedfrom the samples, were fixed in 10% formalin, embedded in paraffin, andsectioned according to standard protocol. Sections were stained with ananti-SGP28 polyclonal antibody (as described above). The results areshown in FIGS. 6A-B. Strong staining was observed in the epithelialcells of the prostate gland, especially at the lumenal borders. Stainingwas also observed within the lumen, indicating high level expression andsecretion of SGP28 in prostate cancer and PIN.

Similarly, polyclonal anti-SGP28 was used to assess the ability todetect SGP28 expression in prostate cancer metastases. FIGS. 7A-B showthe results of immunohistochemical analysis demonstrating SGP28 proteinexpression in prostate cancer metastases to bone (FIG. 7A) and lymphnode (FIG. 7B).

The high expression of SGP28 in prostate cancer and PIN was furtherdemonstrated using immunohistochemistry, and the results are shown inFIGS. 8A-D. FIG. 8A shows immunohistochemical detection of SGP28 inprostate cancer at a magnification of 200×; FIG. 8B shows the same at800×. SGP28 expression in PIN is shown in FIG. 8C at 200× and in FIG. 8Dat 800×. A summary of the results of immunohistochemical analysis isshown in Table 3 for prostate cancer and PIN, and in Table 4 for a widerrange of tissues. The lack of immunohistochemically detectableexpression in the wide range of tissues examined, including normalprostate, combined with strong staining in prostate cancer and PIN,indicates that SGP28 is a particularly suitable target forantibody-based diagnostics, including evaluation of biopsy specimens andin vivo imaging. TABLE 3 Immunohistochemistry Summary for ProstateCancer and PIN Tissue Staining Intensity Normal prostate/BPH  0/14 NonePIN 1/1 Strong Prostate cancer 6/9 Strong Lymph node metastases 4/5Moderate to strong Bone metastatases 2/4 Moderate to strong

TABLE 4 Immunohistochemistry Summary for Human Tissues Stainingintensity Tissue None Prostate (8/8) BPH (6/6) Pancreas Liver LungTestis Colon Spleen Cerebellum Heart Kidney Light Fallopian tubes Coloncancer Salivary glands Moderate to strong PIN (1/1) Prostate cancer(6/9) Lymph node mets (4/5) Bone mets (2/4)

Example 11 Identification of Potential Signal Transduction Pathways

To determine whether SGP28 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing SGP28. Thesetranscriptional reporters contain consensus binding sites for knowntranscription factors which lie downstream of well characterized signaltransduction pathways. The reporters and examples of there associatedtranscription factors, signal transduction pathways, and activationstimuli are listed below.

-   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress-   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation-   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress-   4. ARE-luc, androgen receptor; steroids/MAPK;    growth/differentiation/apoptosis-   5. p53-luc, p⁵³; SAPK; growth/differentiation/apoptosis-   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

SGP28-mediated effects may be assayed in cells showing mRNA expression.Luciferase reporter plasmids may be introduced by lipid mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellsextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Example 12 In Vitro Assays of SGP28 Function

The expression profile of SGP28 in prostate cancer suggests a functionalrole in tumor initiation, progression and/or maintenance. SGP28 functioncan be assessed in mammalian cells using in vitro approaches. Formammalian expression, SGP28 can be cloned into a number of appropriatevectors, including pcDNA 3.1 myc-His-tag and the retroviral vectorpSRαtkneo (Muller et al, 1991, MCB 11:1785). Using such expressionvectors, SGP28 can be expressed in several cancer cell lines, includingfor example PC-3, NIH 3T3, LNCaP and 293T. Expression of SGP28 can bemonitored using anti-SGP28 antibodies.

Mammalian cell lines expressing SGP28 can be tested in several in vitroand in vivo assays, including cell proliferation in tissue culture,activation of apoptotic signals, primary and metastatic tumor formationin SCID mice, and in vitro invasion using a membrane invasion culturesystem (MICS) (Welch et al., Int. J. Cancer 43: 449-457). SGP28 cellphenotype is compared to the phenotype of cells that lack expression ofSGP28. In addition, cells treated with and without exogenously addedSGP28 protein may be analyzed for altered growth parameters.

Cell lines expressing SGP28 can also be assayed for alteration ofinvasive and migratory properties by measuring passage of cells througha matrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and SGP28 overexpressing PC3, 3T3 and LNCaPcells. To assay whether SGP28 has chemoattractant properties, parentalindicator cells are monitored for passage through the porous membranetoward a gradient of SGP28 conditioned media compared to control media.This assay may also be used to qualify and quantify specificneutralization of the SGP28 induced effect by candidate cancertherapeutic compositions.

In order to establish whether SGP28 binds to cellular proteins expressedin prostate cancer cells and other cancer cells or normal cells, twoapproaches may be taken. In the first approach, in vitro assay forrecombinant HIS-tagged SGP28 binding to various cell lines are used. Inanother approach, a recombinant alkaline phosphatase-SGP28 fusionprotein is generated using the AP-TAG system from GenHunter Corporation(Nashville, Tenn., cat# Q202), and the AP-TAG fusion used to test SGP28binding to a variety of prostate cancer cell lines as described (Chengand Flanagan, 1994, Cell 79:157-168). After washing the cells and addingthe AP substrate BCIP, which forms an insoluble blue precipitate upondephosphorylation, SGP28 binding is determined by identifying cellsstaining blue under the light microscope. Various cancer cell lines canbe examined, including without limitation, various prostate cancer celllines (e.g., LNCaP, PC-3, DU145, TSUPR, LAPC4). Other cell lines such asPREC prostate cell line, 293T, PIN cells, and NIH 3T3, etc. may also beexamined. Additionally, the LAPC and other prostate cancer xenograftsmay be tested. Equilibrium dissociation rate constants may be calculatedto evaluate the strength of the binding interaction. In addition, thenumber of cell surface receptors per cell can be determined. Cell linesor tissues with the highest binding capacity for SGP28 would bepreferred for cloning the SGP28 receptor or other binding partner.

In another functional assay, NIH-3T3 cells stably expressing SGP28 canbe analyzed for their ability to form colonies in soft agar. In theseexperiments, cells used in such procedures (e.g. NIH-3T3 cells), can betransfected to stably express SGP28 or neo or activated-Ras (as the testgene, the negative and the positive controls, respectively) in order toassess the transforming capabilities of SGP28. Typically experiments areperformed in duplicate and the assays are evaluated approximately 4weeks after cell plating. Where experimental observations demonstratethat SGP28 induces an increase in colony formation relative to anegative control (e.g. neo) such results indicate that SGP28 hassignificant transforming capabilities.

Example 13 In Vivo Assay for SGP28 Tumor Growth Promotion

The effect of the SGP28 protein on tumor cell growth may be evaluated invivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected SQ on each flank with 1×10⁶ of a prostate cell linecontaining tkNeo empty vector or SGP28. At least two strategies may beused: (1) Constitutive SGP28 expression under regulation of an LTRpromoter, and (2) Regulated expression under control of an induciblevector system, such as ecdysone, tet, etc. Tumor volume is thenmonitored at the appearance of palpable tumors and followed over time todetermine if SGP28 expressing cells grow at a faster rate. Additionally,mice may be implanted with 1×10⁵ of the same cells orthotopically todetermine if SGP28 has an effect on local growth in the target tissue(i.e., prostate) or on the ability of the cells to metastasize,specifically to lungs, lymph nodes, liver, bone marrow, etc. The effectof SGP28 on bone tumor formation and growth may be assessed by injectingprostate tumor cells intratibially, as described in Example 1.

These assays are also useful to determine the SGP28 inhibitory effect ofcandidate therapeutic compositions, such as for example, SGP28antibodies, SGP28 antisense molecules and ribozymes.

Example 14 Functional Assays for Binding of SGP28 to Cells

Several secreted proteins have been described in prostate cancer, anumber of which have been shown to participate in the process of tumorformation and progression (Inoue K., 2000, Clin. Cancer Res. 6:2104-19,Dow J K, deVere White R W, 2000, Urology 55:800-6). As SGP28 is asecreted protein, one of its potential functions is to regulate themicroenvironment of prostate cancer and of metastatic disease. In orderto test this possibility, SGP28 can be expressed and purified as arecombinant protein, such as GST-SGP28 or SGP28-Myc/His. Purifiedrecombinant-SGP28 (whether GST-SGP28 or SGP28-Myc/His) is then incubatedwith a variety of cell types that recapitulate the environment of theprostate, including prostate epithelial cells, prostate tumor celllines, prostate stromal cells, prostate endothelial cells and prostateneuroendocrine cells. In addition, recombinant-SGP28 is also incubatedwith cells found at metastatic sites, such as bone marrow cells andcells of the immune system. Binding of SGP28 to intact cells is detectedby FACS analysis and by calorimetric assay. This analysis is valuable asit identifies with a cell population that binds and may respond toSGP28. In addition, the identification of a target cell populationprovides a means of isolating and identifying SGP28 receptors, therebyproviding additional means of modulating SGP28-mediated events.

Example 15 Assays for Defensin-like Activity of SGP28

SGP28 has a strong homology to defensin proteins, in particular tobeta-defensins. Beta-defensins are secreted products mainly produced byepithelial cells (O'Neil DA et al, 1999, J. Immunol. 163:6718-24;Schroder J M, Harder J., 1999, Int. J. Biochem. Cell. Biol. 31:645-51).Defensins play an important role in preventing infections andsafeguarding the immunity of epithelial tissues. In addition, the humanHNP1 defensin has been shown to induce the death of tumor cells invitro. Investigating the role of SGP28 in cell death, purifiedrecombinant-SGP28 is incubated with a variety of cell types listed aboveand analyzed for apoptotic activity using FACS analysis of Annexin Vstained cells. SGP28 may also function as a chemoattractant, as has beenshown for other defensin molecules (Yang D et al., 2000, Leukoc. Biol.68:9-14; Yang D et al, 1999, Science 286(5439):525-8.). Using achemotactic assay, one can evaluate the effect of SGP28 on the migrationof various types of cells, including epithelial, stromal, endothelialcells as well as monocytes, lymphocytes and dendritic cells.

Example 16 Predicted Binding of SGP28 Peptides to HLA-A2

To identify SGP28 peptides predicted to bind to the human MHC class Imolecule HLA-A2, the complete amino acid sequence of the SGP28 proteinwas entered into the HLA Peptide Motif Search algorithm found in theBioinformatics and Molecular Analysis Section (BIMAS) Web site(http://bimas.dcrt.nih.gov/). The results of SGP28 predicted bindingpeptides are shown in Table 5. The top 10 ranking candidates are shownalong with their location, the amino acid sequence of each specificpeptide, and an estimated binding score. The binding score correspondsto the estimated half-time of dissociation of complexes containing thepeptide at 37° C. at pH 6.5. Peptides with the highest binding score(i.e., 999.9 for SGP28 peptide 2) are predicted to be the most tightlybound to HLA Class I on the cell surface and thus represent the bestimmunogenic targets for T-cell recognition. Actual binding of peptidesto HLA-A2 can be evaluated by stabilization of HLA-A2 expression on theantigen-processing defective cell line T2 (Xue et al., 1997, Prostate30:73-8; Peshwa et al., 1998, Prostate 36:129-38). Immunogenicity ofspecific peptides can be evaluated in vitro by stimulation of CD8+cytotoxic T lymphocytes (CTL) in the presence of dendritic cells (Xue etal., 1997, Prostate 30:73-8; Peshwa et al., 1998, Prostate 36:129-38).TABLE 5 SGP28 Peptides Having Highest Predicted Binding Scores Score(Estimate of half time of Start Subsequence Residue disassoci- RankPosition Listing ation) 1  2-10 TLFPVLLFL (SEQ ID NO: 17) 999.9 2  6-14VLLFLVAGL (SEQ ID NO: 18) 309.1 3 30-38 ALLTTQTQV (SEQ ID NO: 19) 257.34 142-150 VVWYSSYLV (SEQ ID NO: 20) 85.9 5 222-230 TLTCKHQLV (SEQ ID NO:21) 69.6 6 175-183 GNWANRLYV (SEQ ID NO: 22) 20.7 7  7-15 LLFLVAGLL (SEQID NO: 23) 17.5 8 141-149 QVVWYSSYL (SEQ ID NO: 24) 10.3 9 134-142AVVGHYTQV (SEQ ID NO: 25) 9.1 10 211-219 DLYSNCKSL (SEQ ID NO: 26) 5.1

Throughout this application, various publications are referenced. Thedisclosures of these publications are hereby incorporated by referenceherein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1. A method of detecting the presence of a cancer expressing SGP28protein that comprises determining the level of SGP28 protein expressedby cells in a test tissue sample from an individual and comparing thelevel so determined to the level of SGP28 expressed in a correspondingnormal sample, the presence of elevated SGP28 protein in the test samplerelative to the normal sample providing an indication of the presence ofsuch cancer in the individual.
 2. The method of claim 1, whereindetermining the level of SGP28 protein expressed by the cells comprisescontacting the cells with an antibody that specifically binds SGP28protein.
 3. The method of claim 2, wherein the antibody comprises apolyclonal antibody.
 4. The method of claim 2, wherein the antibodycomprises a monoclonal antibody.
 5. A method of monitoring SGP28 geneproducts comprising determining the status of SGP28 gene productsexpressed by cells in a test tissue sample from an individual andcomparing the status so determined to the status of SGP28 gene productsin a corresponding normal sample, the presence of altered status ofSGP28 gene products in the test sample relative to the normal sampleproviding an indication of dysregulated cell growth within theindividual.
 6. A method of examining a biological sample for evidence ofdysregulated cellular growth comprising comparing the status of SGP28 inthe biological sample to the status of SGP28 in a corresponding normalsample, wherein alterations in the status of SGP28 in the biologicalsample are associated with dysregulated cellular growth.
 7. The methodof claim 6, wherein the status of SGP28 in the biological sample isevaluated by examining levels of SGP28 mRNA expression or levels ofSGP28 protein expression.
 8. The method of claim 6, wherein analteration in the status of SGP28 is identified by the presence of SGP28expressing cells in a biological sample from a tissue in which SGP28expressing cells are normally absent.
 9. A method of diagnosing thepresence of cancer in an individual comprising: (a) determining thelevel of SGP28 mRNA expressed in a test sample obtained from theindividual; and (b) comparing the level so determined to the level ofSGP28 mRNA expressed in a comparable known normal tissue sample, thepresence of elevated SGP28 mRNA expression in the test sample relativeto the normal tissue sample providing an indication of the presence ofcancer.
 10. A method of diagnosing the presence of cancer in anindividual comprising: (a) determining the level of SGP28 proteinexpressed in a test sample obtained from the individual; and (b)comparing the level so determined to the level of SGP28 proteinexpressed in a comparable known normal tissue sample, the presence ofelevated SGP28 protein in the test sample relative to the normal tissuesample providing an indication of the presence of cancer.
 11. The methodof claim 10, wherein the cancer is prostate or colon cancer, and thetest and normal tissue samples are selected from the group consisting ofprostate tissue, colon tissue, lymphatic tissue, serum, blood or semen.12. The method of claim 10, wherein determining the level of SGP28protein expressed in the test sample comprises contacting the samplewith an antibody that specifically binds SGP28 protein.
 13. The methodof claim 12, wherein the antibody comprises a polyclonal antibody. 14.The method of claim 12, wherein the antibody comprises a monoclonalantibody. 15-38. (canceled)